Synthetic human neutralizing monoclonal antibodies to human immunodeficiency virus

ABSTRACT

The present invention describes synthetic human monoclonal antibodies that immunoreact with and neutralize human immunodeficiency virus (HIV). The synthetic monoclonal antibodies of this invention exhibit enhanced binding affinity and neutralization ability to gp120. Also disclosed are immunotherapeutic and diagnostic methods of using the monoclonal antibodies, as well as cell lines for producing the monoclonal antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US94/11907, filed Oct. 19, 1994, whichis a continuation-in-part application of U.S. application Ser. No.08/308,841, filed Sep. 19, 1994, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 08/233,619, filed Apr.26, 1994, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 08/139,409, filed Oct. 19, 1993, now abandoned.

This invention was made with government support under Contract No.AI33292 by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The present invention relates generally to the field of immunology andspecifically to synthetic human monoclonal antibodies that bind andneutralize human immunodeficiency virus (HIV).

BACKGROUND

1. HIV Immunotherapy

HIV is the focus of intense studies as it is the causative agent foracquired immunodeficiency syndrome (AIDS). Immunotherapeutic methods areone of several approaches to prevention, cure or remediation of HIVinfection and HIV-induced diseases. Specifically, the use ofneutralizing antibodies in passive immunotherapies is of centralimportance to the present invention.

Passive immunization of HIV-1 infected humans using human seracontaining polyclonal antibodies immunoreactive with HIV has beenreported. See for example, Jackson et al., Lancet, September 17:647-652,(1988); Karpas et al., Proc. Natl. Acad. Sci., USA, 87:7613-7616 (1990).

Numerous groups have reported the preparation of human monoclonalantibodies that neutralize HIV isolates in vitro. The describedantibodies typically have immunospecificities for epitopes on the HIVglycoprotein gp160 or the related glycoproteins gp120 or gp41. See, forexample Karwowska et al., Aids Research and Human Retroviruses,8:1099-1106 (1992); Takeda et al., J. Clin. Invest., 89:1952-1957(1992); Tilley et al., Aids Research and Human Retroviruses, 8:461-467(1992); Laman et al., J. Virol., 66:1823-1831 (1992); Thali et al., J.Virol., 65:6188-6193 (1991); Ho et al., Proc. Natl. Acad. Sci., USA,88:8949-8952 (1991); D'Souza et al., AIDS, 5:1061-1070 (1991); Tilley etal., Res. Virol., 142:247-259 (1991); Broliden et al., Immunol.,73:371-376 (1991); Matour et al., J. Immunol., 146:4325-4332 (1991); andGorny et al., Proc. Natl. Acad. Sci., USA, 88:3238-3242 (1991). For acurrent review of pathogenesis of HIV infection and therapeuticmodalities including the use of passive immunity with anti-HIVantibodies, see Levy, Microbiol. Rev., 57:183-289 (1993).

To date, none of the reported human monoclonal antibodies have beenshown to be effective in passive immunization therapies. Further, asmonoclonal antibodies, they all each react with an individual epitope onthe HIV envelope surface glycoproteins, gp120 or gp160, or against theV3 loop of gp120 or against the envelope transmembrane glycoprotein,gp41. The epitope against which an effective neutralizing antibodyimmunoreacts has not been identified.

There continues to be a need to develop human monoclonal antibodypreparations with significant HIV neutralization activity. In addition,there is a need for monoclonal antibodies immunoreactive with additionaland diverse neutralizing epitopes on HIV gp120. Additional (new) epitopespecificities are required because, upon passive immunization, theadministered patient can produce an immune response against theadministered antibody, thereby inactivating the particular therapeuticantibody.

Furthermore, the well documented ability of HIV to mutate its envelopeglycoprotein structure and thereby alter its reactivity with the immunesystem of an infected host produces variant “field isolates” whichcompromise the utility of individual antibody preparationsimmunoreactive with an individual laboratory strain of HIV. Existingantibody preparations tend to be less potent against primary fieldisolates of HIV than against laboratory strains. Moore et al.,Perspectives in Drug Discovery and Design, 1:235-250 (1993). Inaddition, no reported human monoclonal antibody has been shown to beeffective at neutralizing multiple strains of HIV. Therefore, there alsocontinues to be a need for a human monoclonal antibody with the abilityto neutralize multiple different strains of HIV.

2. Human Monoclonal Antibodies Produced From Combinatorial PhagemidLibraries

The use of filamentous phage display vectors, referred to as phagemids,has been repeatedly shown to allow the efficient preparation of largelibraries of monoclonal antibodies having diverse and novelimmunospecificities. The technology uses a filamentous phage coatprotein membrane anchor domain as a means for linking gene-product andgene during the assembly stage of filamentous phage replication, and hasbeen used for the cloning and expression of antibodies fromcombinatorial libraries. Kang et al., Proc. Natl. Acad. Sci., USA,88:4363-4366 (1991). Combinatorial libraries of antibodies have beenproduced using both the cpVIII membrane anchor (Kang et al., Proc. Natl.Acad. Sci., USA, 88:4363-4366, 1991) and the cpIII membrane anchor.Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991).

The diversity of a filamentous phage-based combinatorial antibodylibrary can be increased by shuffling of the heavy and light chain genes(Kang et al., Proc. Natl. Acad. Sci., USA, 88:11120-11123, 1991), byaltering the CDR3 regions of the cloned heavy chain genes of the library(Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461, 1992), and byintroducing random mutations into the library by error-prone polymerasechain reactions (PCR). Gram et al., Proc. Natl. Acad. Sci., USA,89:3576-3580 (1992).

Filamentous phage display vectors have also been utilized to producehuman monoclonal antibodies immunoreactive with hepatitis B virus (HBV)or HIV antigens. See, for example Zebedee et al., Proc. Natl. Acad.Sci., USA, 89:3175-3179 (1992); and Burton et al., Proc. Natl. Acad.Sci., USA, 88:10134-10137 (1991), respectively. Human monoclonalantibodies displayed on the surface of bacteriophage through the use ofphage vectors, where the antibodies are specific for HIV-1 antigens,gp120 and gp41, have been generated through screening of combinatoriallibraries. The resultant antibodies have been shown to be immunoreactivewith HIV and to neutralize HIV. See, Barbas et al., J. Mol. Biol.,230:812-823 (1993); Williamson et al., Proc. Natl. Acad. Sci., USA,90:4141-4145 (1993); Burton et al., Chem. Immunol., 56:112-126 (1993);and Barbas et al., Proc. Natl. Acad. Sci., USA, 89:9339-9343 (1992).

While the above-described phage display-derived anti-HIV antibodies havebeen shown to neutralize HIV infection, the screened antibodies arerepresentative of the immune repertoire of an immunized or infectedhost. However, the heavy and light chain pairings isolated for theiraffinity for an antigen in vitro are not necessarily paired in vivo.Although the phage display system allows for unique pairing of heavy andlight chains, in many cases affinity selection restores the approximatepairings. Burton et al., Nature, 359:782-783 (1992). While suchimmunized sources or immune priming by natural infection provides usefulantibody libraries for some antigens, it is not always possible toacquire such libraries.

Although anti-HIV-1 neutralizing antibodies have been obtained throughscreening of phage libraries prepared from HIV-1 positive donors, theresultant antibodies are limited in specificity and affinity by theheavy and light chain amino acid residue sequences.

The diversity of a filamentous phage-based combinatorial antibodylibrary, however, can be increased by shuffling of the heavy and lightchain genes obtained from an initial screen of a library (Kang et al.,Proc. Natl. Acad. Sci. USA, 88:11120-11123, 1991). Another approach isto introduce random mutations into the heavy and light chain genes byerror-prone polymerase chain reactions (PCR). Gram et al., Proc. Natl.Acad. Sci., USA, 89:3576-3580, 1992). Mutagenesis of proteins has beenutilized to alter the function, and in some cases the bindingspecificity, of a protein. Typically, the mutagenesis is site-directed,and therefore laborious depending on the systematic choice of mutationto induce in the protein. See, for example Corey et al., J. Amer. Chem.Soc., 114:1784-1790 (1992), in which rat trypsins were modified bysite-directed mutagenesis. More recently, Riechmann et al., Biochem.,32:8848-8855 (1993), described the use of site-directed mutagenesis andphage display techniques prior to screening the randomized library toincrease the affinity of a single-chain Fab fragment specific for thehapten 2-phenyloxazol-5-one.

A preferred approach, in order to more extensively sample the potentialof antibody structure and function, is the preparation of semisyntheticantibodies in the context of phage display. In these molecules, one ormore of the complementarity determining regions (CDR) of the clonedheavy or light chain genes obtained from screening of the library arealtered resulting in new variable domain amino acid residue sequences.Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461 (1992). Unlikeantibodies cloned from a particular donor, semisynthetic antibodies canhave CDR of any size with any sequence, thereby increasing the potentialto obtain antibodies having new specificities and affinities.

BRIEF DESCRIPTION OF THE INVENTION

Synthetic Fab heterodimers specific for HIV-1 glycoproteins havingenhanced affinity, specificity and neutralizing capacities as comparedto the previously characterized antibodies have now been discovered. Thenew synthetic HIV-1-specific Fab heterodimers are obtained through theuse of the synthetic method of randomly mutagenizing the complementaritydetermining regions (CDR) of the heavy and light chain genes encoding arecombinant Fab antibody to produce an antibody that binds to andneutralizes HIV.

The randomly mutagenized neutralizing antibodies define new epitopes onHIV, particularly on HIV glycoprotein gp120, thereby increasing theavailability of new immunotherapeutic human monoclonal antibodies thatexhibit higher affinity binding to the epitope as compared to antibodiesselected from a non-randomized combinatorial library.

The invention provides synthetic human monoclonal antibodies thatneutralize HIV more efficiently than antibodies selected fromnon-randomized combinatorial libraries. Also provided are amino acidsequences which confer the enhanced neutralization function to theantigen binding domain of a monoclonal antibody, and which can be usedimmunogenically to identify other antibodies that specifically bind andneutralize HIV. The synthetic monoclonal antibodies of the inventionfind particular utility as reagents for the diagnosis and immunotherapyof HIV-induced disease.

A major advantage of the monoclonal antibodies of the invention derivesfrom the fact that they are encoded by a human polynucleotide sequence,i.e., they are human antibodies. Thus, in vivo use of the monoclonalantibodies of the invention for diagnosis and immunotherapy ofHIV-induced disease greatly reduces the problems of significant hostimmune response to the passively administered antibodies which is aproblem commonly encountered when monoclonal antibodies of xenogeneic orchimeric derivation are utilized.

Another major advantage of the human monoclonal antibodies of thepresent invention is that the antibodies have dramatically increasedimmunoaffinity for the target antigen, making the antibodiesparticularly potent both diagnostically and therapeutically.

In one embodiment, the invention contemplates a synthetic humanmonoclonal antibody capable of immunoreacting with and neutralizinghuman immunodeficiency virus (HIV). A synthetic monoclonal antibody hasthe capacity to reduce HIV infectivity titer in an in vitro virusinfectivity assay by 50% at a concentration of less than 100 nanograms(ng) of antibody per milliliter (ml) of culture medium in the assay. Inpreferred embodiments, the monoclonal antibody reduces infectivitytiters 50% at a concentration is less than 20 ng/ml, and preferably lessthan 10 ng/ml.

A preferred synthetic monoclonal antibody is a Fab fragment. Morepreferred are synthetic monoclonal antibody molecules that immunoreactwith an HIV glycoprotein, particularly the HIV glycoprotein gp120.

The invention also describes human monoclonal antibodies, and theirmethod of preparation, which exhibit enhanced or improved virusneutralization capacity for multiple different strains of HIV, i.e.,increased breadth of virus strain neutralizing capacity. Thus, apreferred human monoclonal antibody has the ability to neutralize apreselected first HIV strain as described above, and further has thecapacity to reduce the HIV infectivity titer of a second field strain ofHIV in the in vitro virus infectivity assay by 50% at a concentration ofless than 10 micrograms (ug) of antibody per milliliter (ml). Byneutralizing multiple strains of HIV, the present antibodies exhibitstrain crossreactivity and multi-strain neutralizing abilities.

A preferred synthetic human monoclonal antibody has the bindingspecificity of a monoclonal antibody comprising a heavy chainimmunoglobulin variable region amino acid residue sequence selected fromthe group consisting of SEQ ID NOs 2, 3, 4 and 5. Another preferredsynthetic human monoclonal antibody has the binding specificity of amonoclonal antibody comprising a light chain immunoglobulin variableregion amino acid residue sequence in SEQ ID NO 6.

In preferred embodiment, the invention describes the neutralizingantibodies as being immunoreactive with HIV glycoprotein gp120 with adissociation constant (K_(d)) of about 1×10⁻⁸ M or less, preferably fromabout 1×10⁻⁹ M to about 1×10⁻¹¹ M, more preferably from about 1×10⁻¹⁰ Mto about 1×10⁻¹¹ M, and most preferably from about 1×10⁻¹¹ M to about1×10⁻¹² M.

A preferred human monoclonal antibody having the above high-affinitybinding specificity comprises a heavy chain immunoglobulin variableregion amino acid residue sequence selected from the group consisting ofSEQ ID NOs 1, 2, 3, 4, 5, 54, 55, 56, 57, 58, 59, 89, 90, 91 and 92, andconservative substitutions thereof. In addition, a preferred humanmonoclonal antibody having the above high-affinity binding specificitycomprises a light chain immunoglobulin variable region amino acidresidue sequence selected from the group consisting of SEQ ID NOs 6, 69,70, 73, 75, 76, 77, 79, 80, 82, 83, 84, 85, 86, 87 and 88, andconservative substitutions thereof. Particularly preferred is a humanmonoclonal antibody wherein the monoclonal antibody has the bindingspecificity of a monoclonal antibody having heavy and light chainimmunoglobulin variable region amino acid residue sequences in pairsselected from the group consisting of SEQ ID NOs 2:6, 3:6, 4:6, 5:6,3:69, 3:70, 3:73, 3:75, 3:76, 3:77, 3:79, 3:80, 3:82, 3:83, 3:84, 3:85,3:86, 3:87, 54:6, 55:6, 56:6, 57:6, 58:6, 59:6, 89:6, 89:88, 90:86,90:88, 91:6, 91:88 and 92:88, and conservative substitutions thereof.

Also contemplated are methods of producing a synthetic anti-HIVmonoclonal antibody using random mutagenesis methods for sequentiallymutagenizing one or more preselected domains of the immunoglobulin heavychain, preferably a complementarity determining region (CDR), andsubsequently selecting for antibodies which strongly immunoreact withand neutralize HIV.

In another embodiment, the invention describes a polynucleotide sequenceencoding a heavy or light chain immunoglobulin variable region aminoacid residue sequence portion of a synthetic human monoclonal antibodyof this invention. Also contemplated are DNA expression vectorscontaining the polynucleotide, and host cells containing the vectors andpolynucleotides of the invention.

The invention also contemplates a method of detecting humanimmunodeficiency virus (HIV) comprising contacting a sample suspected ofcontaining HIV with a diagnostically effective amount of the syntheticmonoclonal antibody of this invention, and determining whether thesynthetic monoclonal antibody immunoreacts with the sample. The methodcan be practiced in vitro or in vivo, and may include a variety ofmethods for determining the presence of an immunoreaction product.

In another embodiment, the invention describes a method for providingpassive immunotherapy to human immunodeficiency virus (HIV) disease in ahuman, comprising administering to the human an immunotherapeuticallyeffective amount of the synthetic monoclonal antibody of this invention.The administration can be provided prophylactically, and by a parenteraladministration. Pharmaceutical compositions containing one or more ofthe different synthetic human monoclonal antibodies are described foruse in the therapeutic methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 illustrates the amino acid residue sequences of variable heavy(V_(H)) domains of human Fabs binding to gp120. The Fab heterodimerdesignations corresponding to the DNA clone from which the Fabs areexpressed are indicated in the left hand column. The Fabs, MT4, 3b1,3b3, 3b4 and 3b9, have been respectively assigned SEQ ID NOs 1-5 and arelisted as such in the Sequence Listing. Fab MT4 is expressed from theoriginal clone selected from a plasmid library generated from patientMT. The library was screened against gp120 as described in Example 1A.The synthetic human HIV-1 neutralizing Fabs, 3b1, 3b3, 3b4 and 3b9, arethe Fabs that resulted from randomizing CDR1 and CDR3 of the heavy chainas described in Example 1, specifically Examples 1B and 1E. Thesequenced regions of each Fab listed from left to right are frameworkregion 1 (FR1), complementary determining region 1 (CDR1), frameworkregion 2 (FR2), complementary determining region 2 (CDR2), frameworkregion 3 (FR3), complementary determining region 3 (CDR3), and frameworkregion 4 (FR4).

FIG. 2 illustrates the amino acid residue sequence of the variable kappalight (V_(K)) domain of the Fabs binding to gp120. The Fab heterodimerdesignation is indicated in the left hand column. The light chain of FabMT4 has been assigned SEQ ID NO 6 and is listed as such in the SequenceListing. Fab MT4 is expressed from the original clone selected from aplasmid library generated from patient MT. The library was screenedagainst gp120 as described in Example 1A. In addition, since the MT4light chain was not randomized in the generation of the synthetic Fabsdescribed in FIG. 1, the light chain amino acid sequence of MT4 ispresent in each of the Fabs 3b1, 3b3, 3b4 and 3b9, as described inExample 1.

FIG. 3 is a graph that illustrates the correlation of the bindingaffinity and neutralization ability of the synthetic Fabs of thisinvention, 3b1, 3b3, 3b4 and 3b9, with gp120 from the MN strain ofHIV-1. The binding affinity (K_(a) M⁻¹) is plotted on the X-axis asindicated as an exponential value E+ and with the neutralization ability(Neut 50, IC₅₀ M⁻¹) plotted on the Y-axis. The IC₅₀ values, rather thanthe neutralization titer, are plotted for the neutralization data in thegraph. The binding affinity data for generating the graph are presentedin Table 1 in Example 1H under the column heading K_(a) (M⁻¹). Theneutralization data are presented in Table 3 in Example 1H under thecolumn heading IC₅₀ (M⁻¹). The data for each Fab is indicated in thegraph. The relationship between binding affinity and neutralizationability is relatively linear as described in Example 1H with Fab 3b3exhibiting both the highest binding affinity and neutralization ability.All four synthetic Fabs of this invention having randomized CDR1 andCDR3 in the heavy chain exhibited enhanced binding affinity andneutralization ability over the original clone, MT4.

FIG. 4 illustrates the coding nucleotide strand shown in the 5′ to 3′direction of the heavy chain variable domain of original clone pMT4. Thesequence is also listed in SEQ ID NO 7. The corresponding encoded heavychain variable domain amino acid residue sequence is shown in FIG. 1.The pMT4 nucleotide sequence was randomized according to the presentinvention at the region of residues 82-96 corresponding to CDR1 and inseparate mutagenesis procedures from residues 292-303, and then from304-318 in CDR3. The positions of the residues are indicated in thefigure numerically at every tenth residue marked with an asterisk.

FIG. 5 illustrates the amino acid residue sequences of variable heavy(V_(H)) domains of human Fabs binding to gp120. In Experiment A, CDR1 israndomized over amino acid residues 31-35. The CDR1 randomized librarywas screened against gp120 as described in Example 1A, and Example 1D.The amino acid residues 31-35 deduced from the nucleotide sequence of 12selected Fabs in Experiment A is listed from right to left ascomplementary determining region 1 (CDR1). The 12 Fabs have beendesignated SEQ ID NOs 14-25 from the top of the column to the bottom,respectively, and are listed as such in the Sequence Listing. Thesequence of the CDR1 of MT4 from which the other Fabs were derived isdesignated SEQ ID NO 8. The CDR1 randomized library was furtherrandomized in the complementary determining region 3 (CDR3) region asdescribed in Examples 1B and 1E. The deduced amino acid sequence of theCDR1 and CDR3 from eight Fabs selected from the randomized CDR1 and CDR3library are given in Experiment B. The Fab heterodimer designationscorresponding to the DNA clone from which the Fabs are expressed areindicated in the right hand column. The complete heavy chain variabledomain sequences of Fabs, MT4, 3b1, 3b3, 3b4 and 3b9, have beenrespectively assigned SEQ ID NOs 1-5 and are listed as such in theSequence Listing. The CDR1 of the Fabs 3b1, 3b2, 3b3, 3b4, 3b6, 3b7,3b8, and 3b9 have been respectively assigned SEQ ID NOs 26-33 and arelisted as such in the Sequence Listing. Fab MT4 is expressed from theoriginal clone selected from a plasmid library generated from patientMT. The sequence of the CDR3 of MT4 from which the other Fabs werederived is designated SEQ ID NO 34. The sequenced regions of each Fablisted from left to right are amino acid residues 96-99 of CDR3. TheCDR3 of the Fabs 3b1, 3b2, 3b3, 3b4, 3b6, 3b7, 3b8, and 3b9, have beenrespectively assigned SEQ ID NOs 35-42 and are listed as such in theSequence Listing.

FIG. 6 illustrates a schematic representation of the heavy and lightchain expression-control region of the phagemid Fab-display expressionvector, pComb3H, as described in Example 2.

FIGS. 7A and 7B illustrate the amino acid residue sequences of variableheavy (V_(H)) chain domains of gp120-specific human Fabs derived frommutagenizing the heavy chain CDR1 of phagemid MT4-3 (pMT4-3). The H4H1series Fab heterodimer designations corresponding to the DNA clone fromwhich the Fabs are expressed are indicated in the left hand column. Theamino acid residue sequence of the heavy chain variable domain of thetemplate Fab MT4 is presented under the indicated regions as describedin the legend for FIG. 1. The portions of the amino acid residuesequence of the derived Fabs that are identical to the template Fab areindicated by ditto (″) marks. The mutagenized CDR for each derived Fabis shown. The complete variable domain for each derived Fab havingmutagenized heavy chain CDR1 is listed in the Sequence Listingcorresponding to the assigned identifiers. The Fabs were obtained asdescribed in Example 2.

FIG. 8 illustrates the restriction map of the pPho-TT expression vectoras described in Example 2. The complete nucleotide coding sequence ofpPho-TT expression vector in 5′ to 3′ direction containing thenucleotide sequences encoding the heavy and light chain variable domainsof a tetanus toxin (TT)-directed Fab is listed in the Sequence Listingas SEQ ID NO 51. The description of this vector and use thereof toexpress soluble Fabs of this invention replacing the anti-TT Fab isdescribed in Example 2.

FIGS. 9A and 9B illustrate the amino acid residue sequences of variableheavy (V_(H)) chain domains of gp120-specific human Fabs derived frommutagenizing the heavy chain CDR3 of phagemid 3b3 (p3b3). The M556series Fab heterodimer designations corresponding to the DNA clone fromwhich the Fabs are expressed are indicated in the left hand column ofFIG. 9A. The amino acid residue sequence of the heavy chain variabledomain of the template Fab 3b3 are presented under the indicated regionsas described in the legend for FIG. 1. The portions of the amino acidresidue sequence of the derived Fabs that are identical to the templateFab is indicated by ditto (″) marks. The mutagenized CDR for eachderived Fab is shown. The complete variable domain for each derived Fabhaving mutagenized heavy chain CDR3 is listed in the Sequence Listingcorresponding to the assigned identifiers. The Fabs were obtained asdescribed in Example 2.

FIG. 10 illustrates the coding nucleotide strand shown in the 5′ to 3′direction of the light chain variable domain of original clone pMT4-3.The sequence is also listed in SEQ ID NO 62. The corresponding encodedlight chain variable domain amino acid residue sequence is shown in FIG.2 (SEQ ID NO 6).

FIGS. 11A and 11B illustrate the amino acid residue sequences ofvariable kappa light (V_(K)) chain domains of gp120-specific human Fabsderived from mutagenizing the light chain CDR1 of phagemid 3b3 (p3b3)which is the same light chain as that encoded by pMT4-3 (SEQ ID NO 6)shown in FIG. 2. The A-D series Fab heterodimer designationscorresponding to the DNA clone from which the Fabs are expressed areindicated in the left hand column of FIG. 11A. The amino acid residuesequence of the light chain variable domain of the template Fab 3b3 ispresented under the indicated regions as described in the legend forFIG. 1. The portions of the amino acid residue sequence of the derivedFabs that are identical to the template Fab are indicated by ditto (″)marks. The mutagenized CDR for each derived Fab is shown. The completevariable domain for each derived Fab having mutagenized light chain CDR1is listed in the Sequence Listing with the assigned identifiers. TheFabs were obtained as described in Example 2.

FIGS. 12A and 12B illustrate the amino acid residue sequences ofvariable kappa light (V_(K)) chain domains of gp120-specific human Fabsderived from mutagenizing the light chain CDR3 of phagemid 3b3 (p3b3)which is the same light chain as that of pMT4-3 (SEQ ID NO 6) shown inFIG. 2. The H4L3 series Fab heterodimer designations corresponding tothe DNA clone from which the Fabs are expressed are indicated in theleft hand column of FIG. 12A. The amino acid residue sequence of thelight chain variable domain of the template Fab 3b3 is presented underthe indicated regions as described in the legend for FIG. 1. Theportions of the amino acid residue sequence of the derived Fabs that areidentical to the template Fab are indicated by ditto (″) marks. Themutagenized CDR for each derived Fab is shown. The complete variabledomain for each derived Fab having mutagenized light chain CDR3 islisted in the Sequence Listing with the assigned identifiers. The Fabswere obtained as described in Example 2.

FIGS. 13A and 13B illustrate the amino acid residue sequences ofvariable kappa light (V_(K)) chain domains of gp120-specific human Fabsderived from mutagenizing the light chain CDR3 of phagemid D encodingthe previously CDR1-mutagenized and selected Fab D (SEQ ID NO 70) shownin FIGS. 11A, 11B, 13A and 13B. The QA series Fab heterodimerdesignations corresponding to the DNA clone from which the Fabs areexpressed are indicated in the left hand column of FIG. 13A. The aminoacid residue sequence of the light chain variable domain of the templateFab D is presented under the indicated regions as described in thelegend for FIG. 1. The portions of the amino acid residue sequence ofthe derived Fabs that are identical to the template Fab are indicated byditto (″) marks. The mutagenized CDR for each derived Fab is shown. Thecomplete variable domain for each derived Fab having mutagenized lightchain CDR3 is listed in the Sequence Listing with the assignedidentifiers. The Fabs were obtained as described in Example 3.

FIGS. 14A and 14B illustrate the amino acid residue sequence of thevariable kappa light (V_(K)) chain domain of the gp120-specific humancomposite light chain designated L42. The L42 phagemid for encoding theL42 light chain was derived from ligating the Sac I/Kpn I fragment ofphagemid D (encodes Fab D shown in FIGS. 11A, 11B, 13A and 13B) with theKpn I/Xba I fragment of phagemid H4L3-2 (encodes Fab H4L3-2 shown inFIGS. 12A and 12B). The amino acid residue sequence of the L42 compositelight chain variable domain is presented under the indicated regions asdescribed in the legend for FIG. 1 and in the Sequence Listing in SEQ IDNO 88. The L42 light chain was obtained as described in Example 3.

FIGS. 15A and 15B illustrate the amino acid residue sequences of thevariable heavy (V_(H)) chain domains of the gp120-specific humancomposite heavy chains designated H31, H33, H101 and H103. Thecomposites were prepared as described in Example 3. The amino acidresidue sequence of each composite heavy chain variable domain ispresented under the indicated regions as described in the legend forFIG. 1 and in the identified SEQ ID NOs. The portions of the amino acidresidue sequence of the composite heavy chain variable domain regionsthat are identical to that shown for H31 are indicated by ditto (″)marks. The mutagenized CDR1 and CDR3 are separately indicated for eachheavy chain composite.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552-59 (1969) and adopted at 37 CFR §1.822(b)(2)), abbreviationsfor amino acid residues are shown in the following Table ofCorrespondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left-to-right orientation in the conventionaldirection of amino terminus to carboxy terminus. In addition, the phrase“amino acid residue” is broadly defined to include the amino acidslisted in the Table of Correspondence and modified and unusual aminoacids, such as those listed in 37 CFR 1.822(b)(4), and incorporatedherein by reference. Furthermore, it should be noted that a dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or acovalent bond to an amino-terminal group such as NH₂ or acetyl or to acarboxy-terminal group such as COOH.

Recombinant DNA (rDNA) Molecule: A DNA molecule produced by operativelylinking two DNA segments. Thus, a recombinant DNA molecule is a hybridDNA molecule comprising at least two nucleotide sequences not normallyfound together in nature. rDNA's not having a common biological origin,i.e., evolutionarily different, are said to be “heterologous”.

Vector: A rDNA molecule capable of autonomous replication in a cell andto which a DNA segment, e.g., gene or polynucleotide, can be operativelylinked so as to bring about replication of the attached segment. Vectorscapable of directing the expression of genes encoding for one or morepolypeptides are referred to herein as “expression vectors”.Particularly important vectors allow cloning of cDNA (complementary DNA)from mRNAs produced using reverse transcriptase.

Receptor: A receptor is a molecule, such as a protein, glycoprotein andthe like, that can specifically (non-randomly) bind to another molecule.

Antibody: The term antibody in its various grammatical forms is usedherein to refer to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantibody combining site or paratope. Exemplary antibody molecules areintact immunoglobulin molecules, substantially intact immunoglobulinmolecules and portions of an immunoglobulin molecule, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v).

Antibody Combining Site: An antibody combining site is that structuralportion of an antibody molecule comprised of a heavy and light chainvariable and hypervariable regions that specifically binds (immunoreactswith) an antigen. The term immunoreact in its various forms meansspecific binding between an antigenic determinant-containing moleculeand a molecule containing an antibody combining site such as a wholeantibody molecule or a portion thereof.

Monoclonal Antibody: A monoclonal antibody in its various grammaticalforms refers to a population of antibody molecules that contain only onespecies of antibody combining site capable of immunoreacting with aparticular epitope. A monoclonal antibody thus typically displays asingle binding affinity for any epitope with which it immunoreacts. Amonoclonal antibody may therefore contain an antibody molecule having aplurality of antibody combining sites, each immunospecific for adifferent epitope, e.g., a bispecific monoclonal antibody. Althoughhistorically a monoclonal antibody was produced by immortalization of aclonally pure immunoglobulin secreting cell line, a monoclonally purepopulation of antibody molecules can also be prepared by the methods ofthe present invention.

Synthetic Monoclonal Antibody: The term “synthetic” indicates, when usedin the phrase “synthetic monoclonal antibody”, that the antibody is notnaturally isolated, but rather is the product of mutagenesis, asdescribed herein, in the heavy or light chain variable regions of clonedhuman immunoglobulin genes to produce artificial antibodies havingcharacteristic amino acid residue sequences which impart theimmunospecificity, immunoaffinity and HIV-neutralization activity asdescribed herein.

Fusion Polypeptide: A polypeptide comprised of at least two polypeptidesand a linking sequence to operatively link the two polypeptides into onecontinuous polypeptide. The two polypeptides linked in a fusionpolypeptide are typically derived from two independent sources, andtherefore a fusion polypeptide comprises two linked polypeptides notnormally found linked in nature.

Upstream: In the direction opposite to the direction of DNAtranscription, and therefore going from 5′ to 3′ on the non-codingstrand, or 3′ to 5′ on the mRNA.

Downstream: Further along a DNA sequence in the direction of sequencetranscription or read out, that is traveling in a 3′- to 5′-directionalong the non-coding strand of the DNA or 5′- to 3′-direction along theRNA transcript.

Cistron: Sequence of nucleotides in a DNA molecule coding for an aminoacid residue sequence and including upstream and downstream DNAexpression control elements.

Leader Polypeptide: A short length of amino acid sequence at the aminoend of a polypeptide, which carries or directs the polypeptide throughthe inner membrane and so ensures its eventual secretion into theperiplasmic space and perhaps beyond. The leader sequence peptide iscommonly removed before the polypeptide becomes active.

Reading Frame: Particular sequence of contiguous nucleotide triplets(codons) employed in translation. The reading frame depends on thelocation of the translation initiation codon.

B. Synthetic Human Monoclonal Antibodies

The present invention relates to synthetic human monoclonal antibodieswhich are specific for, and neutralize human immunodeficiency virus(HIV). In a preferred embodiment of the invention, human monoclonalantibodies are disclosed which are capable of binding epitopicpolypeptide sequences in an HIV protein, and preferably in HIVglycoprotein gp120 or gp160.

The synthetic monoclonal antibodies are unusual insofar as they are ofhuman derivation, but are modified by recombinant methodologies to yielda synthetic product which exhibits high immunoreaction affinity fortarget antigen. Furthermore, the synthetic monoclonal antibodies have apotent capacity to neutralize HIV. The capacity to neutralize HIV isexpressed as a concentration of antibody molecules required to reducethe infectivity titer of a suspension of HIV when assayed in an typicalin vitro infectivity assay, such as is described herein. A syntheticmonoclonal antibody of this invention has the capacity to reduce HIVinfectivity titer in an in vitro virus infectivity assay by 50% at aconcentration of less than 100 nanograms (ng) of antibody per milliliter(ml) of culture medium in the assay, and preferably reduces infectivitytiters 50% at a concentration of less than 20 ng/ml, and more preferablyat concentrations less than 10 ng/ml.

Exemplary and preferred synthetic monoclonal antibodies described hereinare effective at 5-20 ng/ml, and therefore are particularly well suitedfor inhibiting HIV in vitro and in vivo.

Also disclosed is an antibody having a specified amino acid sequence,which sequence confers the ability to bind a specific epitope and toneutralize HIV when the virus is bound by these antibodies. A humanmonoclonal antibody with a claimed specificity, and like humanmonoclonal antibodies with like specificity, are useful in the diagnosisand immunotherapy of HIV-induced disease.

The term “HIV-induced disease” means any disease caused, directly orindirectly, by HIV. An example of a HIV-induced disease is acquiredautoimmunodeficiency syndrome (AIDS), and any of the numerous conditionsassociated generally with AIDS which are caused by HIV infection.

Thus, in one aspect, the present invention is directed to synthetichuman monoclonal antibodies which are reactive with a HIV neutralizationsite and cell lines which produce such antibodies. The isolation of celllines producing monoclonal antibodies of the invention is described ingreat detail further herein, and can be accomplished using the phagemidvector library methods described herein, and using routine screeningtechniques which permit determination of the elementary immunoreactionand neutralization patterns of the monoclonal antibody of interest.Thus, if a human monoclonal antibody being tested binds and neutralizesHIV, then the human monoclonal antibody being tested and the humanmonoclonal antibody produced by the cell lines of the invention areconsidered equivalent.

It is also possible to determine, without undue experimentation, if ahuman monoclonal antibody has the same (i.e., equivalent) specificity asa human monoclonal antibody of this invention by ascertaining whetherthe former prevents the latter from binding to HIV. If the humanmonoclonal antibody being tested competes with the human monoclonalantibody of the invention, as shown by a decrease in binding by thehuman monoclonal antibody of the invention in standard competitionassays for binding to solid phase gp120 antigen, then it is likely thatthe two monoclonal antibodies bind to the same, or a closely related,epitope.

Still another way to determine whether a human monoclonal antibody hasthe specificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with HIVwith which it is normally reactive, and then add the human monoclonalantibody being tested to determine if the human monoclonal antibodybeing tested is inhibited in its ability to bind HIV. If the humanmonoclonal antibody being tested is inhibited then, in all likelihood,it has the same, or functionally equivalent, epitopic specificity as themonoclonal antibody of the invention. Screening of human monoclonalantibodies of the invention, can be also carried out utilizing HIVneutralization assays and determining whether the monoclonal antibodyneutralizes HIV.

The ability to neutralize HIV at one or more stages of virus infectionis a desirable quality of a human monoclonal antibody of the presentinvention. Virus neutralization can be measured by a variety of in vitroand in vivo methodologies. Exemplary methods described herein fordetermining the capacity for neutralization are the in vitro assays thatmeasure inhibition of HIV-induced syncytia formation, and assays thatmeasure the inhibition of output of core p24 antigen from a cellinfected with HIV.

As shown herein, the immunospecificity of a human monoclonal antibody ofthis invention can be directed to epitopes that are shared acrossserotypes and/or strains of HIV, or can be specific for a single strainof HIV, depending upon the epitope. Thus, a preferred human monoclonalantibody can immunoreact with HIV-1, HIV-2, or both, and can immunoreactwith one or more of the HIV-1 strains IIIB, MN, RF, SF-2, Z2, Z6, CDC4,ELI and the like strains.

In a particularly preferred embodiment, the invention describes numeroushuman monoclonal antibodies produced by the present methods with eachantibody exhibiting the ability to neutralize multiple strains of HIV,particularly field isolates. By sequential randomization of the CDRregions of heavy and/or light chain genes as described herein, multipleantibody species were produced that could neutralize several differentfield strains of HIV.

Thus, the invention also contemplates a human monoclonal antibodycapable of immunoreacting with and neutralizing a first preselectedhuman immunodeficiency virus (HIV), such as the laboratory isolate MN orIIIB, that is further capable of immunoreacting with and neutralizingone or more other (i.e., second) strains of HIV, particularly fieldstrains. In this embodiment, supported by the teachings of the Examples,the antibody has the capacity to reduce HIV infectivity titer in an invitro virus infectivity assay of the first HIV strain by 50% at aconcentration of less than 100 nanograms (ng) of antibody per milliliter(ml), and has the capacity to reduce HIV infectivity titer of a secondfield strain of HIV in the same in vitro virus infectivity assay by 50%at a concentration of less than about 10 micrograms (ug) of antibody permilliliter (ml). In more preferred embodiments and depending upon theparticular HIV strain, the capacity to reduce infectivity titers by 50%can be exhibited at lower antibody concentrations, such as below 1ug/ml, and preferably below 100 ng/ml.

The immunospecificity of an antibody, its HIV-neutralizing capacity, andthe attendant affinity the antibody exhibits for the epitope, aredefined by the epitope with which the antibody immunoreacts. The epitopespecificity is defined at least in part by the amino acid residuesequence of the variable region of the heavy chain of the immunoglobulinthe antibody, and in part by the light chain variable region amino acidresidue sequence. Preferred human monoclonal antibodies immunoreact withglycoprotein gp120.

A preferred human monoclonal antibody of this invention has the bindingspecificity of a monoclonal antibody comprising a heavy chainimmunoglobulin variable region amino acid residue sequence selected fromthe group of sequences consisting of SEQ ID NOs 2, 3, 4 and 5, andconservative substitutions thereof.

Another preferred human monoclonal antibody of this invention has thebinding specificity of a monoclonal antibody having a light chainimmunoglobulin variable region amino acid residue sequence of SEQ ID NO6, and conservative substitutions thereof.

Using known combinatorial library shuffling and screening methods, onecan identify new heavy and light chain pairs (H:L) that function as aHIV-neutralizing monoclonal antibody. In particular, one can shuffle aknown heavy chain, derived from an HIV-neutralizing human monoclonalantibody, with a library of light chains to identify new H:L pairs thatform a functional antibody according to the present invention.Similarly, one can shuffle a known light chain, derived from anHIV-neutralizing human monoclonal antibody, with a library of heavychains to identify new H:L pairs that form a functional antibodyaccording to the present invention.

Particularly preferred human monoclonal antibodies are those having theimmunoreaction (binding) specificity of a monoclonal antibody havingheavy and light chain immunoglobulin variable region amino acid residuesequences in pairs (H:L) selected from the group consisting of SEQ IDNOs 2:6, 3:6, 4:6 and 5:6, and conservative substitutions thereof. Thedesignation of two SEQ ID NOs with a colon, e.g., 2:6, is to connote aH:L pair formed by the heavy and light chain, respectively, amino acidresidue sequences shown in SEQ ID NO 2 and SEQ ID NO 6, respectively.

Particularly preferred is a human monoclonal antibody having the bindingspecificity of the monoclonal antibody having a heavy chain sequenceshown in SEQ ID NOs 2, 3, 4 or 5, and further that contains thesequences present in the antibody molecule MT4 encoded by the pMT4expression vectors deposited with the ATCC on Oct. 19, 1993, asdescribed further herein. By “having the binding specificity” is meantequivalent monoclonal antibodies which exhibit the same or similarimmunoreaction and neutralization properties, and which compete forbinding to an HIV antigen.

In a preferred embodiment, the immunoaffinity of the subject antibody isparticularly high, thereby providing high potency in therapeuticapplications and providing high specificity with low background indiagnostic applications. In this embodiment, a subject synthetic humanmonoclonal antibody, in addition to the above-recited neutralizationcapacity, immunoreacts with HIV with a dissociation constant (K_(d)) ofabout 1×10⁻⁸ molar (M) or less. That is, antibodies which haveaffinities greater than 10⁻⁸ M are particularly preferred. By thepresent synthetic methods, numerous monoclonal antibodies have beengenerated with affinities, expressed as K_(d), in the range of 10⁻⁹ to10⁻¹² M.

Insofar as either the light or heavy chain variable region, or both, canbe modified in sequence by the present methods, a preferred antibody ofthis invention may contain a preferred heavy chain, light chain, orboth. Thus a preferred synthetic human monoclonal antibody has thebinding specificity of a monoclonal antibody comprising a heavy chainimmunoglobulin variable region amino acid residue sequence selected fromthe group consisting of SEQ ID NOs 1, 3, 54, 55, 56, 57, 58, 59, 89, 90,91 and 92, and conservative substitutions thereof. In addition, apreferred synthetic human monoclonal antibody has the bindingspecificity of a monoclonal antibody comprising a light chainimmunoglobulin variable region amino acid residue sequence selected fromthe group consisting of SEQ ID NOs 6, 69, 70, 73, 75, 76, 77, 79, 80,82, 83, 84, 85, 86, 87 and 88, and conservative substitutions thereof.

A particularly preferred synthetic human monoclonal antibody has adissociation constant from about 1×10⁻⁹ M to about 1×10⁻¹⁰ M. In thisembodiment, a specific preferred antibody has the binding specificity ofa monoclonal antibody having heavy and light chain immunoglobulinvariable region amino acid residue sequences in pairs selected from thegroup consisting of SEQ ID NOs 3:6, 3:69, 3:70, 3:73, 3:75, 3:76, 3:77,3:79, 3:80, 3:82, 3:83, 3:84, 3:87, 54:6, 55:6, 56:6, 57:6, 58:6, 59:6,90:88, 91:6, 91:88 and 92:88, and conservative substitutions thereof.

A still more preferred synthetic human monoclonal antibody has adissociation constant from about 1×10⁻¹⁰ M to about 1×10⁻¹¹ M. In thisembodiment, a specific preferred antibody has the binding specificity ofa monoclonal antibody having heavy and light chain immunoglobulinvariable region amino acid residue sequences in pairs selected from thegroup consisting of SEQ ID NOs 3:85, 3:86, 89:6 and 90:86, andconservative substitutions thereof.

A more preferred synthetic human monoclonal antibody has a dissociationconstant from about 1×10⁻¹¹ M to about 1×10⁻¹² M. In this embodiment, aspecific preferred antibody has the binding specificity of a monoclonalantibody having heavy and light chain immunoglobulin variable regionamino acid residue sequences in pairs shown in SEQ ID NOs 89:88, andconservative substitutions thereof. Particularly preferred is anantibody having the binding specificity of the monoclonal antibodyproduced by plasmid pPHO-H31/L42-1 contained in ATCC accession number69691.

Exemplary antibodies having the above immunoaffinities are described inthe examples.

The term “conservative variation” as used herein denotes the replacementof an amino acid residue by another, biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies having thesubstituted polypeptide also neutralize HIV. Analogously, anotherpreferred embodiment of the invention relates to polynucleotides whichencode the above noted heavy and/or light chain polypeptides and topolynucleotide sequences which are complementary to these polynucleotidesequences. Complementary polynucleotide sequences include thosesequences which hybridize to the polynucleotide sequences of theinvention under stringent hybridization conditions.

By using the human monoclonal antibodies of the invention, it is nowpossible to produce anti-idiotypic antibodies which can be used toscreen human monoclonal antibodies to identify whether the antibody hasthe same binding specificity as a human monoclonal antibody of theinvention and also used for active immunization (Herlyn et al., Science,232:100 (1986)). Such anti-idiotypic antibodies can be produced usingwell-known hybridoma techniques (Kohler et al., Nature, 256:495 (1975)).An anti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the human monoclonal antibody produced by thecell line of interest. These determinants are located in thehypervariable region of the antibody. It is this region which binds to agiven epitope and, thus, is responsible for the specificity of theantibody. An anti-idiotypic antibody can be prepared by immunizing ananimal with the monoclonal antibody of interest. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an antibody to these idiotypicdeterminants. By using the anti-idiotypic antibodies of the immunizedanimal, which are specific for the human monoclonal antibody of theinvention produced by a cell line which was used to immunize the secondanimal, it is now possible to identify other clones with the sameidiotype as the antibody of the hybridoma used for immunization.Idiotypic identity between human monoclonal antibodies of two cell linesdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing anti-idiotypic antibodies, it is possible to identify otherhybridomas expressing monoclonal antibodies having the same epitopicspecificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody. Thus, theanti-idiotypic monoclonal antibody can be used for immunization, sincethe anti-idiotype monoclonal antibody binding domain effectively acts asan antigen.

In one preferred embodiment, the invention contemplates a truncatedimmunoglobulin molecule comprising a Fab fragment derived from a humanmonoclonal antibody of this invention. The Fab fragment, lacking Fcreceptor, is soluble, and affords therapeutic advantages in serum halflife, and diagnostic advantages in modes of using the soluble Fabfragment. The preparation of a soluble Fab fragment is generally knownin the immunological arts and can be accomplished by a variety ofmethods. A preferred method of producing a soluble Fab fragment isdescribed herein.

Human monoclonal antibodies offer particular advantages over murinemonoclonal antibodies, particularly insofar as they can be usedtherapeutically in humans. Specifically, human antibodies are notcleared from the circulation as rapidly as “foreign” antigens, and donot activate the immune system in the same manner as foreign antigensand foreign antibodies.

The invention also contemplates, in one embodiment, a monoclonalantibody of this invention produced by the present methods.

C. Immunotherapeutic Methods and Compositions

The synthetic human monoclonal antibodies of this invention can also beused immunotherapeutically for HIV disease due to their demonstratedneutralization activity and high immunoaffinity for target antigen. Theterm “immunotherapeutically” or “immunotherapy” as used herein inconjunction with the monoclonal antibodies of the invention denotes bothprophylactic as well as therapeutic administration. Thus, the monoclonalantibodies can be administered to high-risk patients in order to lessenthe likelihood and/or severity of HIV-induced disease, administered topatients already evidencing active HIV infection, or administered topatients at risk of HIV infection.

1. Therapeutic Compositions

The present invention therefore contemplates therapeutic compositionsuseful for practicing the therapeutic methods described herein.Therapeutic compositions of the present invention contain aphysiologically tolerable carrier together with at least one species ofhuman monoclonal antibody as described herein, dissolved or dispersedtherein as an active ingredient. In a preferred embodiment, thetherapeutic composition is not immunogenic when administered to a humanpatient for therapeutic purposes, unless that purpose is to induce animmune response, as described elsewhere herein.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a human without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as sterile injectableseither as liquid solutions or suspensions, aqueous or non-aqueous,however, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared. The preparation can also beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, propylene glycon,polyethylene glycol and other solutes.

Liquid compositions can also contain liquid phases in addition to waterand to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, organicesters such as ethyl oleate, and water-oil emulsions.

A therapeutic composition contains an HIV-neutralizing of a humanmonoclonal antibody of the present invention, typically an amount of atleast 0.1 weight percent of antibody per weight of total therapeuticcomposition. A weight percent is a ratio by weight of antibody to totalcomposition. Thus, for example, 0.1 weight percent is 0.1 grams ofantibody per 100 grams of total composition.

2. Therapeutic Methods

In view of the demonstrated HIV neutralizing ability of the humanmonoclonal antibodies of the present invention, the present disclosureprovides for a method for neutralizing HIV in vitro or in vivo. Themethod comprises contacting a sample believed to contain HIV with acomposition comprising a therapeutically effective amount of a humanmonoclonal antibody of this invention. A preferred therapeuticallyeffective amount is an amount sufficient to effect a 50% reduction ininfectivity, preferably a 90% reduction, and more preferably a 99%reduction when assayed in an in vitro assay as described herein.

For in vivo modalities, the method comprises administering to thepatient a therapeutically effective amount of a physiologicallytolerable composition containing a human monoclonal antibody of theinvention. Thus, the present invention describes in one embodiment amethod for providing passive immunotherapy to HIV disease in a humancomprising administering to the human an immunotherapeutically effectiveamount of the monoclonal antibody of this invention.

A representative patient for practicing the present passiveimmunotherapeutic methods is any human exhibiting symptoms ofHIV-induced disease, including AIDS or related conditions believed to becaused by HIV infection, and humans at risk of HIV infection, i.e.,prophylactic treatments to prevent infection. Patients at risk ofinfection by HIV include babies of HIV-infected pregnant mothers,recipients of transfusions known to contain HIV, users of HIVcontaminated needles, individuals who have participated in high risksexual activities with known HIV-infected individuals, and the like risksituations.

In one embodiment, the passive immunization method comprisesadministering a composition comprising more than one species of humanmonoclonal antibody of this invention, preferably directed tonon-competing epitopes or directed to distinct serotypes or strains ofHIV, as to afford increased effectiveness of the passive immunotherapy.

A therapeutically (immunotherapeutically) effective amount of a humanmonoclonal antibody is a predetermined amount calculated to achieve thedesired effect, i.e., to neutralize the HIV present in the sample or inthe patient, and thereby decrease the amount of detectable HIV in thesample or patient. In the case of in vivo therapies, an effective amountcan be measured by improvements in one or more symptoms associated withHIV-induced disease occurring in the patient, or by serologicaldecreases in HIV antigens.

Thus, the dosage ranges for the administration of the monoclonalantibodies of the invention are those large enough to produce thedesired effect in which the symptoms of the HIV disease are amelioratedor the likelihood of infection decreased. The dosage should not be solarge as to cause adverse side effects, such as hyperviscositysyndromes, pulmonary edema, congestive heart failure, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art.

The dosage can be adjusted by the individual physician in the event ofany complication.

A therapeutically effective amount of an antibody of this invention istypically an amount of antibody such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration from about 0.1 microgram (ug) per milliliter (ml) to about100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, and usuallyabout 5 ug/ml. Stated differently, the dosage can vary from about 0.1mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one ormore dose administrations daily, for one or several days.

The human monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual infusion over time. Although theHIV infection is typically systemic and therefore most often treated byintravenous administration of therapeutic compositions, other tissuesand delivery means are contemplated where there is a likelihood that thetissue targeted contains infectious HIV. Thus, human monoclonalantibodies of the invention can be administered intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity,transdermally, and can be delivered by peristaltic means.

The therapeutic compositions containing a human monoclonal antibody ofthis invention are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

As an aid to the administration of effective amounts of a monoclonalantibody, a diagnostic method for detecting a monoclonal antibody in thesubject's blood is useful to characterize the fate of the administeredtherapeutic composition.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the human monoclonal antibodies ofthe invention, the medicament being used for immunotherapy of HIVdisease.

D. Diagnostic Assay Methods

The present invention also contemplates various assay methods fordetermining the presence, and preferably amount, of HIV in a sample suchas a biological fluid or tissue sample using a human monoclonal antibodyof this invention as an immunochemical reagent to form an immunoreactionproduct whose amount relates, either directly or indirectly, to theamount of HIV in the sample.

Those skilled in the art will understand that there are numerous wellknown clinical diagnostic chemistry procedures in which animmunochemical reagent of this invention can be used to form animmunoreaction product, in vitro or in vivo, whose amount relates to theamount of HIV present in a body sample. Thus, while exemplary assaymethods are described herein, the invention is not so limited.

Various heterogenous and homogeneous protocols, either competitive ornoncompetitive, can be employed in performing an assay method of thisinvention. Examples of types of immunoassays which can utilizemonoclonal antibodies of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA) and thesandwich (immunometric) assay. Detection of the antigens using themonoclonal antibodies of the invention can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of HIV. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbio-luminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibodies of theinvention, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to themonoclonal antibodies of the invention can be done using standardtechniques common to those of ordinary skill in the art.

For purposes of the invention, HIV may be detected by the monoclonalantibodies of the invention when present in samples of biological fluidsand tissues. Any sample containing a detectable amount of HIV can beused. A sample can be a liquid such as urine, saliva, cerebrospinalfluid, blood, serum and the like, or a solid or semi-solid such astissues, feces, and the like, or, alternatively, a solid tissue such asthose commonly used in histological diagnosis.

Another labeling technique which may result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use haptens such as biotin, whichreacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, whichcan react with specific anti-hapten antibodies.

The monoclonal antibodies of the invention are suited for use in vitro,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier for the detection of HIV insamples, as described above. The monoclonal antibodies in theseimmunoassays can be detectably labeled in various ways for in vitro use.

In using the human monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled human monoclonalantibody is given in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledhuman monoclonal antibody is administered in sufficient quantity toenable detection of the site having the HIV antigen for which themonoclonal antibodies are specific.

The concentration of detectably labeled human monoclonal antibody whichis administered should be sufficient such that the binding to HIV isdetectable compared to the background. Further, it is desirable that thedetectably labeled monoclonal antibody be rapidly cleared from thecirculatory system in order to give the best target-to-background signalratio.

As a rule, the dosage of detectably labeled human monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. The dosage of human monoclonalantibody can vary from about 0.01 mg/m² to about 500 mg/m², preferably0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m² to about10 mg/m². Such dosages may vary, for example, depending on whethermultiple injections are given, tissue, and other factors known to thoseof skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹Tl.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵M, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The human monoclonal antibodies of the invention can be used in vitroand in vivo to monitor the course of HIV disease therapy. Thus, forexample, by measuring the increase or decrease in the number of cellsinfected with HIV or changes in the concentration of HIV present in thebody or in various body fluids, it would be possible to determinewhether a particular therapeutic regimen aimed at ameliorating the HIVdisease is effective.

E. Diagnostic Systems

The present invention also describes a diagnostic system, preferably inkit form, for assaying for the presence of HIV in a sample according tothe diagnostic methods described herein. A diagnostic system includes,in an amount sufficient to perform at least one assay, a subject humanmonoclonal antibody, as a separately packaged reagent.

In another embodiment, a diagnostic system is contemplated for assayingfor the presence of an anti-HIV monoclonal antibody in a body fluidsample such as for monitoring the fate of therapeutically administeredantibody. The system includes, in an amount sufficient for at least oneassay, a subject antibody as a control reagent, and preferably apreselected amount of HIV antigen, each as separately packagedimmunochemical reagents.

Instructions for use of the packaged reagent are also typicallyincluded.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

In embodiments for detecting HIV in a body fluid, a diagnostic system ofthe present invention can include a label or indicating means capable ofsignaling the formation of an immunocomplex containing a humanmonoclonal antibody of the present invention.

The word “complex” as used herein refers to the product of a specificbinding reaction such as an antibody-antigen reaction. Exemplarycomplexes are immunoreaction products.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents. Such labels are themselves wellknown in clinical diagnostic chemistry and constitute a part of thisinvention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-amino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabelling agent is aradioactive element that produces gamma ray emissions. Elements whichthemselves emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Crrepresent one class of gamma ray emission-producing radioactive elementindicating groups. Particularly preferred is ¹²⁵I. Another group ofuseful labeling means are those elements such as ¹¹C, ¹⁸F, ¹⁵O and ¹³Nwhich themselves emit positrons. The positrons so emitted produce gammarays upon encounters with electrons present in the animal's body. Alsouseful is a beta emitter.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an “ELISA”format to detect the quantity of an APC inhibitor of this invention in avascular fluid sample such as blood, serum, or plasma. “ELISA” refers toan enzyme-linked immunosorbent assay that employs an antibody or antigenbound to a solid phase and an enzyme-antigen or enzyme-antibodyconjugate to detect and quantify the amount of an antigen present in asample. A description of the ELISA technique is found in Chapter 22 ofthe 4th Edition of Basic and Clinical Immunology by D. P. Sites et al.,published by Lange Medical Publications of Los Altos, Calif. in 1982 andin U.S. Pat. No. 3,654,090; No. 3,850,752; and No. 4,016,043, which areall incorporated herein by reference.

Thus, in some embodiments, a human monoclonal antibody of the presentinvention can be affixed to a solid matrix to form a solid support thatcomprises a package in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from anaqueous medium although other modes of affixation applicable to proteinsand polypeptides well known to those skilled in the art, can be used.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include the cross-linked dextran available under thetrademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.);agarose; beads of polystyrene beads about 1 micron to about 5millimeters in diameter available from Abbott Laboratories of NorthChicago, Ill.; polyvinyl chloride, polystyrene, cross-linkedpolyacrylamide, nitrocellulose- or nylon-based webs such as sheets,strips or paddles; or tubes, plates or the wells of a microtiter platesuch as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems.

The term “package” refers to a solid matrix or material such as glass,plastic (e.g., polyethylene, polypropylene and polycarbonate), paper,foil and the like capable of holding within fixed limits a diagnosticreagent such as a monoclonal antibody of the present invention. Thus,for example, a package can be a bottle, vial, plastic and plastic-foillaminated envelope or the like container used to contain a contemplateddiagnostic reagent or it can be a microtiter plate well to whichmicrogram quantities of a contemplated diagnostic reagent have beenoperatively affixed, i.e., linked so as to be capable of beingimmunologically bound by an antibody or polypeptide to be detected.

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise a humanmonoclonal antibody of the invention which is, or can be, detectablylabelled. The kit may also have containers containing any of the otherabove-recited immunochemical reagents used to practice the diagnosticmethods.

F Methods for Producing a Synthetic HIV-Neutralizing Human MonoclonalAntibody

The present invention describes methods for producing novel syntheticHIV-neutralizing human monoclonal antibodies. The methods are basedgenerally on the use of combinatorial libraries of antibody moleculeswhich can be produced from a variety of sources, and include naivelibraries, modified libraries, and libraries produced directly fromhuman donors exhibiting an HIV-specific immune response.

The combinatorial library production and manipulation methods have beenextensively described in the literature, and will not be reviewed indetail herein, except for those feature required to make and use uniqueembodiments of the present invention. However, the methods generallyinvolve the use of a filamentous phage (phagemid) surface expressionvector system for cloning and expressing antibody species of thelibrary. Various phagemid cloning systems to produce combinatoriallibraries have been described by others. See, for example thepreparation of combinatorial antibody libraries on phagemids asdescribed by Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366(1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991);Zebedee et al., Proc. Natl. Acad. Sci., USA, 89:3175-3179 (1992); Kanget al., Proc. Natl. Acad. Sci., USA, 88:11120-11123 (1991); Barbas etal., Proc. Natl. Acad. Sci., USA, 89:4457-4461 (1992); and Gram et al.,Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992), the disclosures ofwhich are hereby incorporated by reference.

In one embodiment, the method involves first preparing a phagemidlibrary of human monoclonal antibodies by using donor immune cellmessenger RNA from HIV-infected donors. The donors can be symptomatic ofAIDS, but in preferred embodiments the donor is asymptomatic, as theresulting library contains a substantially higher number ofHIV-neutralizing human monoclonal antibodies.

In another embodiment, the donor is naive relative to an immune responseto HIV, i.e., the donor is not HIV-infected. Alternatively, the startinglibrary can be synthetic, or can be derived from a donor who has animmune response to other antigens.

The method for producing a phagemid library of human monoclonalantibodies generally involves (1) preparing separate H and Lchain-encoding gene libraries in cloning vectors using humanimmunoglobulin genes as a source for the libraries, (2) combining the Hand L chain encoding gene libraries into a single dicistronic expressionvector capable of expressing and assembling a heterodimeric antibodymolecule, (3) expressing the assembled heterodimeric antibody moleculeon the surface of a filamentous phage particle, (4) isolating thesurface-expressed phage particle using immunoaffinity techniques such aspanning of phage particles against a preselected antigen, therebyisolating one or more species of phagemid containing particular H and Lchain-encoding genes forming antibody molecules that immunoreact withthe preselected antigen.

As described herein the Examples, the resulting phagemid library is thenmanipulated to increase and/or alter the immunospecificities of themonoclonal antibodies of the library to produce and subsequentlyidentify additional, desirable, human monoclonal antibodies of thepresent invention which are referred to as “synthetic” antibodiesbecause they are not obtained directly from humans, but are derived fromhuman antibodies following synthetic manipulations. Alternatively, aparticular preselected monoclonal antibody can be manipulated to yield asynthetic antibody having superior properties as described furtherherein.

For example, the heavy (H) chain and light (L) chain immunoglobulinmolecule encoding genes can be randomly mixed (shuffled) to create newHL pairs in an assembled immunoglobulin molecule. Additionally, eitheror both the H and L chain encoding genes can be mutagenized in one (ormore) complementarity determining region (CDR) of the variable region ofthe immunoglobulin polypeptide, and subsequently screened for desirableimmunoreaction and neutralization capabilities.

In one embodiment, the H and L genes can be cloned into separate,monocistronic expression vectors, referred to as a “binary” systemdescribed further herein. In this method, step (2) above differs in thatthe combining of H and L chain encoding genes occurs by theco-introduction of the two binary plasmids into a single host cell forexpression and assembly of a phagemid having the surface accessibleantibody heterodimer molecule.

In the present methods, the antibody molecules are monoclonal becausethe cloning methods allow for the preparation of clonally pure speciesof antibody producing cell lines. In addition, the monoclonal antibodiesare human because the H and L chain encoding genes are derived fromhuman immunoglobulin producing immune cells, such as those obtained fromspleen, thymus, bone marrow, and the like.

The method of producing a HIV-neutralizing human monoclonal antibodyalso requires that the resulting antibody library, immunoreactive with apreselected HIV antigen, is screened for the presence of antibodyspecies which have the capacity to neutralize HIV in one or more of theassays described herein for determining neutralization capacity. Thus, apreferred library of antibody molecules is first produced which binds toan HIV antigen, preferably gp160, gp120, the V3 loop region of gp160, orthe CD4 binding site of gp120, and then is screened for the presence ofHIV-neutralizing antibodies as described herein.

Additional libraries can be screened from shuffled libraries foradditional HIV-immunoreactive and neutralizing human monoclonalantibodies.

As a further characterization of the present invention the nucleotideand corresponding amino acid residue sequence of the antibody molecule'sH or L chain encoding gene is determined by nucleic acid sequencing. Theprimary amino acid residue sequence information provides essentialinformation regarding the antibody molecule's epitope reactivity.

Synthetic human monoclonal antibodies of this invention are produced byaltering the nucleotide sequence of a polynucleotide sequence thatencodes a heavy or light chain of a monoclonal antibody. For example, bysite-directed mutagenesis, one can alter the nucleotide sequence of anexpression vector and thereby introduce changes in the resultingexpressed amino acid residue sequence. Thus one can take the amino acidresidue sequence of SEQ ID NO 2, for example, and convert it into theamino acid residue sequence of SEQ ID NO 3 via mutagenesis of thecorresponding nucleic acids. Similarly, one can take a knownpolynucleotide and randomly alter it by random mutagenesis, reintroducethe altered polynucleotide into an expression system and subsequentlyscreen the product H:L pair for HIV-neutralizing activity.

Site-directed and random mutagenesis methods are well known in thepolynucleotide arts, and are not to be construed as limiting as methodsfor altering the nucleotide sequence of a subject polynucleotide.

Due to the presence of the phage particle in an immunoaffinity isolatedantibody, one embodiment involves the manipulation of the resultingcloned genes to truncate the immunoglobulin-coding gene such that asoluble Fab fragment is secreted by the host E. coli cell containing thephagemid vector. Thus, the resulting manipulated cloned immunoglobulingenes produce a soluble Fab which can be readily characterized in ELISAassays for epitope binding studies, in competition assays with knownanti-HIV antibody molecules, and in HIV neutralization assays. Thesolubilized Fab provides a reproducible and comparable antibodypreparation for comparative and characterization studies.

The preparation of soluble Fab is generally described in theimmunological arts, and can be conducted as described herein in theexamples, or as described by Burton et al., Proc. Natl. Acad. Sci., USA,88:10134-10137 (1991).

In addition, one can readily produce a whole antibody molecule thatincludes a functional Fc domain by further engineering of the Fab to addback the polypeptide sequences that define Fc, as is well known.

1. Phase Display Expression Vectors and Polynucleotides for ExpressingAnti-HIV Monoclonal Antibodies

The preparation of human monoclonal antibodies of this inventiondepends, in one embodiment, on the cloning and expression vectors usedto prepare the combinatorial antibody libraries described herein. Thecloned immunoglobulin heavy and light chain genes can be shuttledbetween lambda vectors, phagemid vectors and plasmid vectors at variousstages of the methods described herein.

The phagemid vectors produce fusion proteins that are expressed on thesurface of an assembled filamentous phage particle. The use of phagedisplay vectors allow a particular advantage by providing a means toscreen a very large population of expressed display proteins and therebylocate one or more specific clones that code for a desired bindingreactivity. The use of phage display also facilitates the rapid andreproducible isolation of multiple species of the invention. Forexample, four antibodies were produced in one example of the presentinvention.

The use of phage display vectors derives from the previously describeduse of combinatorial libraries of antibody molecules based on phagemids.The combinatorial library production and manipulation methods have beenextensively described in the literature, and will not be reviewed indetail herein, except for those features required to make and use uniqueembodiments of the present invention. However, the methods generallyinvolve the use of a filamentous phage (phagemid) surface expressionvector system for cloning and expressing antibody species of thelibrary. Various phagemid cloning systems to produce combinatoriallibraries have been described by others. See, for example thepreparation of combinatorial antibody libraries on phagemids asdescribed by Rang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366(1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991);Zebedee et al., Proc. Natl. Acad. Sci., USA, 89:3175-3179 (1992); Kanget al., Proc. Natl. Acad. Sci., USA, 88:11120-11123 (1991); Barbas etal., Proc. Natl. Acad. Sci., USA, 89:4457-4461 (1992); and Gram et al.,Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992), the disclosures ofwhich are hereby incorporated by reference.

A phagemid vector of the present invention is a recombinant DNA (rDNA)molecule containing a nucleotide sequence that codes for and is capableof expressing a fusion polypeptide containing, in the direction ofamino- to carboxy-terminus, (1) a prokaryotic secretion signal domain,(2) a heterologous polypeptide defining an immunoglobulin heavy or lightchain variable region, and (3) a filamentous phage membrane anchordomain. The vector includes DNA expression control sequences forexpressing the fusion polypeptide, preferably prokaryotic controlsequences.

The filamentous phage membrane anchor is preferably a domain of thecpIII or cpVIII coat protein capable of associating with the matrix of afilamentous phage particle, thereby incorporating the fusion polypeptideonto the phage surface.

Preferred membrane anchors for the vector are obtainable fromfilamentous phage M13, f1, fd, and equivalent filamentous phage.Preferred membrane anchor domains are found in the coat proteins encodedby gene III and gene VIII. The membrane anchor domain of a filamentousphage coat protein is a portion of the carboxy terminal region of thecoat protein and includes a region of hydrophobic amino acid residuesfor spanning a lipid bilayer membrane, and a region of charged aminoacid residues normally found at the cytoplasmic face of the membrane andextending away from the membrane.

In the phage f1, gene VIII coat protein's membrane spanning regioncomprises residue Trp-26 through Lys-40, and the cytoplasmic regioncomprises the carboxy-terminal 11 residues from 41 to 52 (Ohkawa et al.,J. Biol. Chem., 256:9951-9958, 1981). An exemplary membrane anchor wouldconsist of residues 26 to 40 of cpVIII. Thus, the amino acid residuesequence of a preferred membrane anchor domain is derived from the M13filamentous phage gene VIII coat protein (also designated cpVIII or CP8). Gene VIII coat protein is present on a mature filamentous phage overthe majority of the phage particle with typically about 2500 to 3000copies of the coat protein.

In addition, the amino acid residue sequence of another preferredmembrane anchor domain is derived from the M13 filamentous phage geneIII coat protein (also designated cpIII). Gene III coat protein ispresent on a mature filamentous phage at one end of the phage particlewith typically about 4 to 6 copies of the coat protein.

For detailed descriptions of the structure of filamentous phageparticles, their coat proteins and particle assembly, see the reviews byRached et al., Microbiol. Rev., 50:401-427 (1986); and Model et al., in“The Bacteriophages: Vol. 2”, R. Calendar, ed. Plenum Publishing Co.,pp. 375-456 (1988).

The secretion signal is a leader peptide domain of a protein thattargets the protein to the periplasmic membrane of gram negativebacteria. A preferred secretion signal is a pelB secretion signal. Thepredicted amino acid residue sequences of the secretion signal domainfrom two pelB gene product variants from Erwinia carotova are describedin Lei et al., Nature, 331:543-546 (1988).

The leader sequence of the pelB protein has previously been used as asecretion signal for fusion proteins (Better et al., Science,240:1041-1043 (1988); Sastry et al., Proc. Natl. Acad. Sci., USA,86:5728-5732 (1989); and Mullinax et al., Proc. Natl. Acad. Sci., USA,87:8095-8099 (1990)).

Another preferred secretion signal is an ompA secretion signal. Thepredicted amino acid residue sequences of the secretion signal domainfrom the ompA gene product from E. coli is described in Movva, et al.,J. Mol. Biol., 147:317-328 (1980).

The leader sequence of the ompA protein has previously been used as asecretion signal for fusion proteins by Skerra et al., Science,240:1038-1041 (1988).

Amino acid residue sequences for other secretion signal polypeptidedomains from E. coli useful in this invention as described in Oliver,Escherichia coli and Salmonella Typhimurium, Neidhard, F. C. (ed.),American Society for Microbiology, Washington, D.C., 1:56-69 (1987).

DNA expression control sequences comprise a set of DNA expressionsignals for expressing a structural gene product and include both 5′ and3′ elements, as is well known, operatively linked to the cistron suchthat the cistron is able to express a structural gene product. The 5′control sequences define a promoter for initiating transcription and aribosome binding site operatively linked at the 5′ terminus of theupstream translatable DNA sequence.

The 3′ control sequences define at least one termination (stop) codon inframe with and operatively linked to the heterologous fusionpolypeptide.

In preferred embodiments, the vector utilized includes a prokaryoticorigin of replication or replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extra chromosomally in a prokaryotic host cell,such as a bacterial host cell, transformed therewith. Such origins ofreplication are well known in the art. Preferred origins of replicationare those that are efficient in the host organism. A preferred host cellis E. coli. For use of a vector in E. coli, a preferred origin ofreplication is ColE1 found in pBR322 and a variety of other commonplasmids. Also preferred is the p15A origin of replication found onpACYC and its derivatives. The ColE1 and p15A replicon have beenextensively utilized in molecular biology, are available on a variety ofplasmids and are described at least by Sambrook et al., in “MolecularCloning: a Laboratory Manual”, 2nd edition, Cold Spring HarborLaboratory Press (1989).

The ColE1 and p15A replicons are particularly preferred for use in oneembodiment of the present invention where two “binary” plasmids areutilized because they each have the ability to direct the replication ofplasmid in E. coli while the other replicon is present in a secondplasmid in the same E. coli cell. In other words, ColE1 and p15A arenon-interfering replicons that allow the maintenance of two plasmids inthe same host (see, for example, Sambrook et al., in “Molecular Cloning:a Laboratory Manual”, 2nd edition, Cold Spring Harbor Laboratory Press(1989), at pages 1.3-1.4). This feature is particularly important whenusing binary vectors because a single host cell permissive for phagereplication must support the independent and simultaneous replication oftwo separate vectors, for example when a first vector expresses a heavychain polypeptide and a second vector expresses a light chainpolypeptide.

In addition, those embodiments that include a prokaryotic replicon canalso include a gene whose expression confers a selective advantage, suchas drug resistance, to a bacterial host transformed therewith. Typicalbacterial drug resistance genes are those that confer resistance toampicillin, tetracycline, neomycin/kanamycin or chloramphenicol. Vectorstypically also contain convenient restriction sites for insertion oftranslatable DNA sequences. Exemplary vectors are the plasmids pUC8,pUC9, pBR322, and pBR329 available from BioRad Laboratories, (Richmond,Calif.) and pPL and pKK223 available from Pharmacia, (Piscataway, N.J.).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. Preferred vectorsare those capable of autonomous replication and expression of structuralgene products present in the DNA segments to which they are operativelylinked. Vectors, therefore, preferably contain the replicons andselectable markers described earlier.

As used herein with regard to DNA sequences or segments, the phrase“operatively linked” means the sequences or segments have beencovalently joined, preferably by conventional phosphodiester bonds, intoone strand of DNA, whether in single or double stranded form. The choiceof vector to which a transcription unit or a cassette of this inventionis operatively linked depends directly, as is well known in the art, onthe functional properties desired, e.g., vector replication and proteinexpression, and the host cell to be transformed, these being limitationsinherent in the art of constructing recombinant DNA molecules.

In a preferred embodiment, a DNA expression vector is designed forconvenient manipulation in the form of a filamentous phage particleencapsulating a genome according to the teachings of the presentinvention. In this embodiment, a DNA expression vector further containsa nucleotide sequence that defines a filamentous phage origin ofreplication such that the vector, upon presentation of the appropriategenetic complementation, can replicate as a filamentous phage in singlestranded replicative form and be packaged into filamentous phageparticles. This feature provides the ability of the DNA expressionvector to be packaged into phage particles for subsequent segregation ofthe particle, and vector contained therein, away from other particlesthat comprise a population of phage particles.

A filamentous phage origin of replication is a region of the phagegenome, as is well known, that defines sites for initiation ofreplication, termination of replication and packaging of the replicativeform produced by replication (see, for example, Rasched et al.,Microbiol. Rev., 50:401-427, 1986; and Horiuchi, J. Mol. Biol.,188:215-223, 1986). A preferred filamentous phage origin of replicationfor use in the present invention is an M13, f1 or fd phage origin ofreplication (Short et al., Nucl. Acids Res., 16:7583-7600, 1988).

Preferred DNA expression vectors for cloning and expressing a phagemiddisplay protein of this invention are the dicistronic plasmid expressionvectors pComb3, pComb3H-TT, pPHO-TT and pMT4-3 described herein. Thecomplete nucleotide sequence of pComb3H-TT and pPHO-TT are shown in theSequence Listing at SEQ ID NOs 43 and 51, respectively.

It is to be understood that, due to the genetic code and its attendantredundancies, numerous polynucleotide sequences can be designed thatencode a contemplated heavy or light chain immunoglobulin variableregion amino acid residue sequence. Thus, the invention contemplatessuch alternate polynucleotide sequences incorporating the features ofthe redundancy of the genetic code.

The phagemid and expression vectors of the present invention can beprepared in a variety of ways. The vectors can be assembled fromcomponent parts using the disclosed complete nucleotide sequences ofpreferred vectors, or can be derived by selective modifications ofexisting vectors. Other methods are apparent to one skilled in the art,and therefore, the methods for preparing and manipulating the vectorsare not to be considered as limiting to the invention.

Insofar as the expression vector for producing a human monoclonalantibody of this invention is carried in a host cell compatible withexpression of the antibody, the invention contemplates a host cellcontaining a vector or polynucleotide of this invention. A preferredhost cell is E. coli, as described herein.

A preferred expression vector plasmid pMT4-3 used to produce a phagemiddisplay protein of this invention was deposited in the form of pMT4 onOct. 19, 1993, pursuant to Budapest Treaty requirements with theAmerican Type Culture Collection (ATCC), Rockville, Md., as describedherein.

The term “pMT4-3” refers to a particular phagemid expression vector inwhich the gene 3 membrane anchor is present, whereas the term “pMT4”refers to the same vector except that the gene 3 membrane anchor hasbeen removed such that the expressed Fab is soluble as described in theExamples.

Insofar as polynucleotides are component parts of a DNA expressionvector for producing a human monoclonal antibody heavy or light chainimmunoglobulin variable region amino acid residue sequence, theinvention also contemplates isolated polynucleotides that encode suchheavy or light chain sequences.

2. Oligonucleotides

The modification of a cloned immunoglobulin to form a synthetic humanantibody molecule of this invention involves the use of syntheticoligonucleotides designed to introduce random mutations into preselecteddomains of the immunoglobulin variable regions of the clonedimmunoglobulin gene. Furthermore, the oligonucleotide strategiesdescribed herein have particular advantages in creating in a singlereaction an extremely large population of different binding sites by theuse of degenerate oligonucleotides.

The general structure of a preferred oligonucleotide for use in one ofthe present methods is described further hereinbelow.

Oligonucleotides for use in the present invention can be synthesized bya variety of chemistries as is well known. An excellent review is“Oligonucleotide Synthesis: A Practical Approach”, ed. M. J. Gait, JRLPress, New York, N.Y. (1990). Suitable synthetic methods include, forexample, the phosphotriester or phosphodiester methods see Narang etal., Meth. Enzymol., 68:90, (1979); U.S. Pat. No. 4,356,270; and Brownet al., Meth. Enzymol., 68:109, (1979). Purification of synthesizedoligonucleotides for use in primer extension and PCR reactions is wellknown. See, example Ausubel et al., “Current Protocols in MolecularBiology”, John Wiley & Sons, New York, (1987). Exemplary synthesis isdescribed in the Examples.

3. Primer Extension Reactions

The term “polynucleotide” as used herein in reference to primers, probesand nucleic acid fragments or segments to be synthesized by primerextension is defined as a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than 3. Itsexact size will depend on many factors, which in turn depends on theultimate conditions of use.

The term “primer” as used herein refers to a polynucleotide whetherpurified from a nucleic acid restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofnucleic acid synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is induced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase, reverse transcriptase and thelike, and at a suitable temperature and pH. The primer is preferablysingle stranded for maximum efficiency, but may alternatively be indouble stranded form. If double stranded, the primer is first treated toseparate it from its complementary strand before being used to prepareextension products. Preferably, the primer is a polydeoxyribonucleotide.The primer must be sufficiently long to prime the synthesis of extensionproducts in the presence of the agents for polymerization. The exactlengths of the primers will depend on many factors, includingtemperature and the source of primer. For example, depending on thecomplexity of the target sequence, a polynucleotide primer typicallycontains 15 to 25 or more nucleotides, although it can contain fewernucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with template.

The primers used herein are selected to be “substantially” complementaryto the different strands of each specific sequence to be synthesized oramplified. This means that the primer must be sufficiently complementaryto non-randomly hybridize with its respective template strand.Therefore, the primer sequence may or may not reflect the exact sequenceof the template. For example, a non-complementary nucleotide fragmentcan be attached to the 5′ end of the primer, with the remainder of theprimer sequence being substantially complementary to the strand. Suchnon-complementary fragments typically code for an endonucleaserestriction site. Alternatively, non-complementary bases or longersequences can be interspersed into the primer, provided the primersequence has sufficient complementarity with the sequence of the strandto be synthesized or amplified to non-randomly hybridize therewith andthereby form an extension product under polynucleotide synthesizingconditions.

Primers of the present invention may also contain a DNA-dependent RNApolymerase promoter sequence or its complement. See for example, Krieget al., Nuc. Acids Res., 12:7057-70 (1984); Studier et al., J. Mol.Biol., 189:113-130 (1986); and Molecular Cloning: A Laboratory Manual,Second Edition, Sambrook et al., eds., Cold Spring Harbor, N.Y. (1989).

When a primer containing a DNA-dependent RNA polymerase promoter isused, the primer is hybridized to the polynucleotide strand to beamplified and the second polynucleotide strand of the DNA-dependent RNApolymerase promoter is completed using an inducing agent such as E. coliDNA polymerase I, or the Klenow fragment of E. coli DNA polymerase. Thestarting polynucleotide is amplified by alternating between theproduction of an RNA polynucleotide and DNA polynucleotide.

Primers may also contain a template sequence or replication initiationsite for a RNA-directed RNA polymerase. Typical RNA-directed RNApolymerase include the QB replicase described by Lizardi et al.,Biotechnology, 6:1197-1202 (1988). RNA-directed polymerases producelarge numbers of RNA strands from a small number of template RNA strandsthat contain a template sequence or replication initiation site. Thesepolymerases typically give a one million-fold amplification of thetemplate strand as has been described by Kramer et al., J. Mol. Biol.,89:719-736 (1974).

The choice of a primer's nucleotide sequence depends on factors such asthe distance on the nucleic acid from the region of the display proteingene into which a binding site is being introduced, its hybridizationsite on the nucleic acid relative to any second primer to be used, andthe like.

The PCR reaction is performed using any suitable method. Generally itoccurs in a buffered aqueous solution, i.e., a PCR buffer, preferably ata pH of 7-9, most preferably about 8. Preferably, a molar excess of theprimer is admixed to the buffer containing the template strand. A largemolar excess of about 10⁴:1 of primer to template is preferred toimprove the efficiency of the process.

The PCR buffer also contains the deoxyribonucleotide triphosphates DATP,dCTP, dGTP, and dTTP and a polymerase, typically thermostable, all inadequate amounts for primer extension (polynucleotide synthesis)reaction. The resulting solution (PCR admixture) is heated to about 90degrees Celsius (90 C.) to 100 C. for about 1 to 10 minutes, preferablyfrom 1 to 4 minutes. After this heating period the solution is allowedto cool to 54 C., which is preferable for primer hybridization. Thesynthesis reaction may occur at from room temperature up to atemperature above which the polymerase (inducing agent) no longerfunctions efficiently. Thus, for example, if DNA polymerase is used asinducing agent, the temperature is generally no greater than about 40 C.An exemplary PCR buffer comprises the following: 50 millimolar (mM) KCl;10 mM Tris-HCl; pH 8.3; 1.5 mM MgCl₂; 0.001% (wt/vol) gelatin, 200micromolar (uM) DATP; 200 uM dTTP; 200 uM dCTP; 200 uM dGTP; and 2.5units Thermus aquaticus DNA polymerase I (U.S. Pat. No. 4,889,818) per100 micriliters (ul) of buffer.

The inducing agent may be any compound or system which will function toaccomplish the synthesis of primer extension products, includingenzymes. Suitable enzymes for this purpose include, for example, E. coliDNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNApolymerase, other available DNA polymerases, reverse transcriptase, andother enzymes, including heat-stable enzymes, which will facilitatecombination of the nucleotides in the proper manner to form the primerextension products which are complementary to each nucleic acid strand.Generally, the synthesis will be initiated at the 3′ end of each primerand proceed in the 5′ direction along the template strand, untilsynthesis terminates, producing molecules of different lengths. Theremay be inducing agents, however, which initiate synthesis at the 5′ endand proceed in the above direction, using the same process as describedabove.

The inducing agent also may be a compound or system which will functionto accomplish the synthesis of RNA primer extension products, includingenzymes. In preferred embodiments, the inducing agent may be aDNA-dependent RNA polymerase such as T7 RNA polymerase, T3 RNApolymerase or SP6 RNA polymerase. These polymerases produce acomplementary RNA polynucleotide. The high turn over rate of the RNApolymerase amplifies the starting polynucleotide as has been describedby Chamberlin et al., The Enzymes, ed. P. Boyer, PP. 87-108, AcademicPress, New York (1982). Another advantage of T7 RNA polymerase is thatmutations can be introduced into the polynucleotide synthesis byreplacing a portion of cDNA with one or more mutagenicoligodeoxynucleotides (polynucleotides) and transcribing thepartially-mismatched template directly as has been previously describedby Joyce et al., Nuc. Acids Res., 17:711-722 (1989). Amplificationsystems based on transcription have been described by Gingeras et al.,in PCR Protocols. A Guide to Methods and Applications, pp 245-252,Academic Press, Inc., San Diego, Calif. (1990).

If the inducing agent is a DNA-dependent RNA polymerase and thereforeincorporates ribonucleotide triphosphates, sufficient amounts of ATP,CTP, GTP and UTP are admixed to the primer extension reaction admixtureand the resulting solution is treated as described above.

The newly synthesized strand and its complementary nucleic acid strandform a double-stranded molecule which can be used in the succeedingsteps of the process, as is known for PCR.

PCR is typically carried out by thermocycling i.e., repeatedlyincreasing and decreasing the temperature of a PCR reaction admixturewithin a temperature range whose lower limit is about 10 C. to about 40C. and whose upper limit is about 90 C. to about 100 C. The increasingand decreasing can be continuous, but is preferably phasic with timeperiods of relative temperature stability at each of temperaturesfavoring polynucleotide synthesis, denaturation and hybridization.

PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in severaltexts including “PCR Technology: Principles and Applications for DNAAmplification”, H. Erlich, ed., Stockton Press, New York (1989); and“PCR Protocols: A Guide to Methods and Applications”, Innis et al.,eds., Academic Press, San Diego, Calif. (1990), the teachings of whichare hereby incorporated by reference.

Preferred PCR reactions using the oligonucleotides and methods of thisinvention are described in the Examples.

4. Methods for Producing a Synthetic Antibody

The present invention provides methods for changing the diversity of amonoclonal antibody or a library of monoclonal antibodies of thisinvention. These methods generally increase the diversity of thelibrary, thereby increasing the pool of possible epitope-bindingcomplexes from which to screen for a desired and improved binding andHIV-neutralizing activity. Alternatively, the methods can be directed atenriching for a class of epitope-binding complexes. The class istypically defined by the ability to bind a particular epitope or familyof epitopes present on a preselected antigen or group of antigens.

The method for producing a synthetic monoclonal antibody generallyinvolves (1) introducing by mutagenesis random mutations into apreselected portion of the immunoglobulin variable gene encoded in aphagemid display protein vector by primer extension with anoligonucleotide as described herein, to form a large population ofdisplay vectors each capable of expressing different monoclonalantibodies displayed on a phagemid surface display protein, (2)expressing the display protein and antibody on the surface of afilamentous phage particle, and (3) isolating the surface-expressedphage particle using affinity techniques such as panning of phageparticles against a preselected target molecule, thereby isolating oneor more species of phagemid containing a synthetic monoclonal antibodythat binds a preselected target molecule.

An exemplary preparation of a binding site in the CDR3 region of a heavychain of an immunoglobulin is described in the Examples. The isolationof a particular vector capable of expressing a binding site of interestinvolves the introduction of the dicistronic expression vector into ahost cell permissive for expression of filamentous phage genes and theassembly of phage particles. Typically, the host is E. coli. Thereafter,a helper phage genome is introduced into the host cell containing thephagemid expression vector to provide the genetic complementationnecessary to allow phage particles to be assembled. The resulting hostcell is cultured to allow the introduced phage genes and display proteingenes to be expressed, and for phage particles to be assembled and shedfrom the host cell. The shed phage particles are then harvested(collected) from the host cell culture media and screened for desirablebinding properties. Typically, the harvested particles are “panned” forbinding with a preselected molecule. The strongly binding particles arethen collected, and individual species of particles are clonallyisolated and further screened for binding to the target molecule and forHIV neutralization. Phage which produce antibody molecules of desiredbinding specificity and neutralization capacity are selected.

As a further characterization of the present invention, the nucleotideand corresponding amino acid residue sequence of the gene coding thebinding site is determined by nucleic acid sequencing. The primary aminoacid residue sequence information provides essential informationregarding the binding site's reactivity.

Mutation of nucleic acid can be conducted by a variety of means, but ismost conveniently conducted in a PCR reaction during a PCR process ofthe present invention. PCR mutagenesis can be random or directed tospecific nucleotide sequences, as is generally well known. ConductingPCR under conditions favorable to random mutagenesis has been describedpreviously, and is referred to as “error prone PCR”. Similarly, directedmutagenesis involves the use of PCR primers designed to target aspecific type of mutation into a specific region of nucleotide sequence.

In one embodiment, the invention contemplates increasing diversity ofone or more epitope-binding complexes by PCR-directed mutation of acomplementarity determining region (CDR) of an antibody variable domainpresent in an epitope-binding complex polypeptide of this invention. CDRmutagenesis has been previously described in general terms for“humanizing” an antibody by introducing human sequences into the CDRregion of a murine antibody. See European Application No. EP 239400.

Thus the invention contemplates a mutagenesis method for altering theimmunological specificity of a cloned immunoglobulin gene present in aDNA vector of this invention. The method provides directed mutagenesisin a preselected CDR of an immunoglobulin gene which comprisessubjecting a recombinant DNA molecule (rDNA) containing the clonedimmunoglobulin gene having a target CDR to PCR conditions suitable foramplifying a preselected region of the CDR. In the method, the rDNAmolecule is subjected to PCR conditions that include a PCR primeroligonucleotide as described below constituting the first primer in aPCR primer pair as is well known to produce an amplified PCR productthat is derived from the preselected CDR but that includes thenucleotide sequences of the PCR primer. The second oligonucleotide inthe PCR amplifying conditions can be any PCR primer derived from theimmunoglobulin gene to be mutagenized, as described herein.

Preferred are methods using an oligonucleotide of this invention asdescribed below.

In a related embodiment, therefore, an oligonucleotide is contemplatedthat is useful as a primer in a polymerase chain reaction (PCR) forinducing mutagenesis in a complementarity determining region (CDR) of animmunoglobulin gene. The oligonucleotide has 3′ and 5′ termini andcomprises (1) a nucleotide sequence at its 3′ terminus capable ofhybridizing to a first framework region of an immunoglobulin gene, (2) anucleotide sequence at its 5′ terminus capable of hybridizing to asecond framework region of an immunoglobulin gene, and (3) a nucleotidesequence between the 3′ and 5′ termini adapted for introducing mutationsduring a PCR into the CDR between the first and second framework regionsof the immunoglobulin gene, thereby mutagenizing the CDR.

Insofar as immunoglobulin genes have three CDR regions on both the heavychain and the light chain of an immunoglobulin, each separated by adistinctive framework region, it is to be understood that the aboveexample is readily applicable to introducing mutations into a specificCDR by selection of the above 5′ and 3′ nucleotide sequences as tohybridize to the framework regions flanking the targeted CDR. Thus theabove first and second framework sequences can be the conservedsequences flanking CDR1, CDR2 or CDR3 on either the heavy or lightchain.

The length of the 3′ and 5′ terminal nucleotide sequences of a subjectmutagenizing oligonucleotide can vary in length as is well known, solong as the length provides a stretch of nucleotides complementary tothe target framework sequences as to hybridize thereto. In the case ofthe 3′ terminal nucleotide sequence, it must be of sufficient length andcomplementarity to the target framework region located 3′ to the CDR tobe mutagenized as to hybridize and provide a 3′ hydroxyl terminus forinitiating a primer extension reaction. In the case of the 5′ terminalnucleotide sequence, it must be of sufficient length and complementarityto the target framework region located 5′ to the CDR to be mutagenizedas to provide a means for hybridizing in a PCR overlap extensionreaction as described above to assemble the complete immunoglobulinheavy or light chain.

Framework regions flanking a CDR are well characterized in theimmunological arts, and include known nucleotide sequences or consensussequences as described elsewhere herein. Where a single, preselectedimmunoglobulin gene is to be mutagenized, the framework-definedsequences flanking a particular CDR are known, or can be readilydetermined by nucleotide sequencing protocols. Where a repertoire ofimmunoglobulin genes are to be mutagenized, the framework-derivedsequences are preferably conserved, as described elsewhere herein.

Preferably, the length of the 3′ and 5′ terminal nucleotide sequencesare each at least 6 nucleotides in length, and can be up to 50 or morenucleotides in length, although these lengths are unnecessary to assureaccurate and reproducible hybridization. Preferred are lengths in therange of 12 to 30 nucleotides, and typically are about 18 nucleotides.

The nucleotide sequence located between the 3′ and 5′ termini adaptedfor mutagenizing a CDR can be any nucleotide sequence, insofar as thenovel sequence will be incorporated by the above methods. However, thepresent approach provides a means to produce a large population ofmutagenized CDR's in a single PCR reaction by the use of a population ofredundant sequences defining randomized or nearly randomized nucleotidesin the CDR to be mutagenized.

A preferred oligonucleotide for mutagenizing CDR1, for example,comprises a nucleotide sequence represented by the formula in thedirection of 5′ to 3′: A-B-C, where A and C represent nucleic acidsequences complementary to FR1 and FR2, respectively, B represents anucleic acid sequence having the formula: [NNS]_(n), wherein N canindependently be any nucleotide, where S is G or C, n is 3 to about 24,and where FR1 and FR2 are the framework regions flanking CDR1 on the 5′and 3′ termini, respectively. Preferably, n is 5.

Similarly, a preferred oligonucleotide for mutagenizing CDR3, forexample, comprises an nucleotide sequence complementary to the sense(coding) strand of CDR3 represented by the formula in the direction of5′ to 3′: C-D-A, where A and C represent nucleic acid sequencescomplementary to FR3 and FR4, respectively, D represents a nucleic acidsequence having the formula: [MNN]_(n), wherein N can independently beany nucleotide, where M is C or A, n is 3 to 24, and where FR3 and FR4are the framework regions flanking CDR3 on the 5′ and 3′ termini,respectively. Preferably, n is 4.

Thus, the invention contemplates a method for increasing the diversityof a library of filamentous phage particles comprising the steps of: a)providing one or more filamentous phage particles according to thepresent invention, and b) mutating the immunoglobulin variabledomain-coding nucleotide sequence present in each provided phageparticle having a DNA expression vector to form a library of phageparticles each containing a mutated immunoglobulin variable domainnucleotide sequence.

The providing can include manipulating the genomes of the phageparticles in the library in order to isolate the nucleic acids inpreparation for a mutagenizing PCR reaction. Manipulations of a phagelibrary to isolate the phage genome for use in a PCR reaction isdescribed elsewhere herein.

Following, mutagenesis of a CDR in a preselected portion to form alibrary of phage containing synthetic monoclonal antibodies, theinvention involves manipulations to change the diversity of the libraryby enriching the library for a preselected class of epitope-bindingcomplexes. The process generally involves affinity selection of thosephage particles in a library that are capable of binding a preselectedantigen. The process of affinity selection, or panning, is described indetail in the Examples.

In a related embodiment, the invention contemplates a method forchanging the diversity of a library of filamentous phage particlescomprising the steps of a) providing a library of filamentous phageparticles according to the present invention, b) contacting the providedlibrary with a preselected ligand under conditions sufficient formembers of the library to bind to the ligand and form a ligand-phageparticle complex, and c) isolating phage particles in the complex awayfrom non-bound library members to form a ligand-enriched librarycomprising phage particles having binding specificity for thepreselected ligand.

In preferred embodiments, the preselected ligand is affixed to a solidsupport, and the ligand-phage particle complex is formed in the solidphase. This embodiment further comprises the steps of: i) washing thesolid support after the contacting step to rinse non-bound librarymembers from the solid support; and ii) eluting any solid-phase boundphage particles off of the solid support. The eluted phage particles arecollected, thereby forming isolated phage particles that comprise anenriched library.

Elution can be conducted under a variety of conditions that disrupt theligand-epitope-binding complex interaction. Typical conditions includehigh salt or low pH buffers. Particularly preferred are buffers of aboutpH 1 to 5, preferably about pH 2 to 3. Alternatively, the interactioncan be disrupted by competition with an excess amount of the preselectedligand in the elution buffer. Both elution procedures are described inthe Examples.

A related embodiment combines the features of both increasing diversityof a library by mutation and enriching the library by panning to“mature” epitope-binding complex affinities for a preselected ligand.Thus it is possible to evolve new binding specificities, and more potentbinding specificities, using the present methods for changing librarydiversity.

The combination of these methods can be configured in a variety of ways,as will be apparent to a skilled practitioner. For example, one canisolate a library, mutagenize (diversify), and then screen (enrich) fora particular binding activity. Alternatively, one can enrich for aparticular activity from a library, mutagenize the specificepitope-binding complex and further enrich the library produced by themutagenesis.

In another permutation on this theme, one can utilize the differencesbetween libraries based on cpIII- and cpVIII-derived membrane anchorsdue to their inherent differences in valency. Because a library of phagehaving the cpIII-derived membrane anchor will typically contain only 1to 4 copies of the epitope-binding complex on the surface of each phageparticle, the phage presents a binding complex of relatively “low”valency, approaching one. In contrast, a library of phage having acpVIII-derived membrane anchor will typically contain 20 to 1000 copiesof the epitope-binding complex on the surface of each phage particle,the particle presents a relatively “high” valency. Thus, cpIII-basedlibraries are referred to as monovalent and cpVIII-based libraries arereferred to as multivalent.

Applying the well-known principles of antibody affinity and valence andthe methods herein, it is demonstrated that a cpIII-based library can beproduced and/or enriched upon screening to contain antibodies withgenerally higher affinity binding interactions, expressed asdissociation binding constants (K_(d)), of 10⁶ to 10¹² M⁻¹, as comparedto the broader range of affinities (binding constants of 10⁴ to 10⁹ M⁻¹)isolatable conventionally or by using a multivalent reagent found in thecpVIII-based library according to the present invention. Therefore, acpVIII-based library is useful to isolate a broad range of affinities ofepitope-binding complexes from low to high, whereas a cpIII-basedlibrary is useful to isolate a narrower range of higher affinityepitope-binding complexes. The high affinity antibodies are particularlypreferred for their strong immunoreactivity and attendant selectivityand strong neutralization abilities as demonstrated herein. Preferredantibodies have affinities of at least 10⁻⁹, preferably at least 10⁻¹⁰,more preferably at least 10⁻¹¹, and most preferably at least 10⁻¹².

The invention also contemplates producing a first enriched library byenrichment of a cpVIII-based library. Thereafter the genes for encodingthe epitope-binding complex polypeptides are transferred into acpIII-based vector, and subsequently enriched for a high affinitybinding interaction. In one embodiment, a mutation step can be utilizedprior to the transfer into the cpIII-based vector.

Thus, the present invention also contemplates a method for maturing theaffinity of an epitope-binding complex encoded by a filamentous phage ofthis invention comprising the steps of: a) providing the genome of afilamentous phage; b) mutating the immunoglobulin variable domain-codingnucleotide sequence present in the provided genome to form a library ofphage particles containing a mutated immunoglobin variable domainnucleotide sequence; c) contacting the library formed in step (b) with apreselected ligand under conditions sufficient for members of thelibrary to bind to the ligand and form a ligand-phage particle complex;and d) isolating phage particles in said complex away from non-boundlibrary members to form a ligand-enriched library comprising phageparticles having binding specificity for the preselected ligand.

In a particularly preferred embodiment demonstrated herein, multiple CDRregions are mutagenized through a series of cycles of mutagenesis andenrichment to synthetically evolve a highly superior monoclonalantibody.

For example, an anti-HIV glycoprotein gp120 monoclonal antibody in theform of a phage display protein was first randomly mutagenized in theCDR1 domain to form a first library of phagemids having syntheticantibodies, and then the library was enriched by panning against apreselected ligand, i.e. HIV gp120 in the solid phase. Thereafter, thehighest affinity binding phagemids in the library were collected, andone or more were selected for further random mutagenesis in the CDR3domain to form a second library of phagemids having syntheticantibodies, and the resulting library was then enriched by panningagainst the preselected ligand gp120 to form a high affinity phagemidshaving synthetic monoclonal antibodies capable of high affinity bindingto HIV gp120. The resulting high affinity antibodies are then screenedin conventional virus neutralization assays described herein to identifythe synthetic antibodies with the highest neutralizing capacity, andselected as an antibody of this invention.

The sequence of mutagenesis and panning events can be altered. Forexample, one can mutagenize CDR3 prior to CDR1, or vice versa.Alternatively, one can further screen the first library forneutralization as a refinement of the enrichment step prior to thesecond mutagenesis and enrichment step.

Thus, in one embodiment, the invention describes a method for producinga synthetic human anti-HIV monoclonal antibody comprising the steps of:

a) providing the genome of filamentous phage encoding a monoclonalantibody having immunoglobulin heavy and light chain variable domains,said heavy chain variable domain present as a fusion polypeptidecontaining a filamentous phage membrane anchor domain, wherein saidmonoclonal antibody immunoreacts with HIV;

b) mutating the immunoglobulin heavy chain variable domain-codingnucleotide sequence present in the provided genome to form a firstlibrary of mutagenized phage particles containing a mutatedimmunoglobulin heavy chain variable domain nucleotide sequence;

c) contacting the library formed in step (b) with a preselected ligandunder conditions sufficient for members of the library to bind to theligand and form a first ligand-phage particle complex;

d) isolating phage particles in said first complex away from non-boundlibrary members to form a first ligand-enriched library comprising phageparticles having binding specificity for said preselected ligand;

e) providing the genome of filamentous phage from said firstligand-enriched library;

f) mutating the immunoglobulin heavy chain variable domain-codingnucleotide sequence present in the provided genome to form a secondlibrary of mutagenized phage particles containing a mutatedimmunoglobulin heavy chain variable domain nucleotide sequence;

g) contacting the library formed in step (f) with a preselected ligandunder conditions sufficient for members of the library to bind to theligand and form a second ligand-phage particle complex; and

h) isolating phage particles in said second complex away from non-boundlibrary members to form a second ligand-enriched library comprisingphage particles having binding specificity for said preselected ligand.

In preferred embodiments, the mutating in steps (b) and (f) are directedto the same region of the immunoglobulin heavy chain variable domain.Alternatively, the mutating in steps (b) and (f) can be directed to twodifferent regions of the immunoglobulin heavy chain variable domain. Forexample, in a preferred method, the mutating in step (b) is directed toa first CDR and the mutating in step (f) is directed to a second CDR. Inpreferred and exemplary methods, the first and second CDR's are CDR1 andCDR3, respectively.

As applied to HIV, it is particularly preferred where the monoclonalantibody of step (a) immunoreacts with HIV glycoprotein gp120 and wherethe preselected ligand used in the enrichment steps (c) and (g) is HIVglycoprotein gp120.

The particularly preferred method for inducing mutagenesis in step (b)comprises inducing mutagenesis in a CDR of an immunoglobulin gene in thephagemid genome which comprises amplifying a portion of the CDR of theimmunoglobulin gene by polymerase chain reaction (PCR) using a PCRprimer oligonucleotide, where the oligonucleotide has 5′ and 3′ terminiand comprises:

a) a nucleotide sequence at the 5′ terminus capable of hybridizing to aframework region upstream of the CDR;

b) a nucleotide sequence at the 3′ terminus capable of hybridizing to aframework region downstream of the CDR; and

c) a nucleotide sequence between the 5′ and 3′ termini according to theformula:

[NNS]_(n),

wherein N is independently any nucleotide, S is G or C, or analogsthereof, and n is 3 to about 24, the 3′ and 5′ terminal nucleotidesequences have a length of about 6 to 50 nucleotides, and sequencescomplementary thereto. Particularly preferred and exemplary of thisembodiment is the method where n is 5, CDR is CDR1, and the upstream anddownstream framework regions are FR1 and FR2, respectively.

Also preferred are methods for inducing mutagenesis in step (f) thatcomprise inducing mutagenesis in a CDR of an immunoglobulin gene in thephagemid genome which comprises amplifying a portion of the CDR of theimmunoglobulin gene by polymerase chain reaction (PCR) using a PCRprimer oligonucleotide, where the oligonucleotide has 5′ and 3′ terminiand comprises:

a) a nucleotide sequence at the 5′ terminus capable of hybridizing tothe antisense (noncoding) framework region downstream of the CDR;

b) a nucleotide sequence at the 3′ terminus capable of hybridizing tothe antisense (noncoding) framework region upstream of the CDR; and

c) a nucleotide sequence between the 5′ and 3′ termini according to theformula:

[MNN]_(n),

wherein N is independently any nucleotide, M is C or A, or analogsthereof, and n is 3 to about 24, the 3′ and 5′ terminal nucleotidesequences have a length of about 6 to 50 nucleotides, and sequencescomplementary thereto. Particularly preferred and exemplary of thisembodiment is the method where n is 4, CDR is CDR3, and the upstream anddownstream framework regions are FR3 and FR4, respectively.

Also contemplated are synthetic monoclonal antibodies immunoreactivewith HIV according to the present invention and produced by anembodiment of the above described processes.

EXAMPLES

The following examples relating to this invention are illustrative andshould not, of course, be construed as specifically limiting theinvention. Moreover, such variations of the invention, now known orlater developed, which would be within the purview of one skilled in theart are to be considered to fall within the scope of the presentinvention hereinafter claimed.

1. Preparation of Synthetic Human Fab Heterodimers That Exhibit EnhancedAffinity to gp120 of HIV-1 and Have Increased Neutralizing Ability

A. Description of nMT4 and an Overview of the Methods to Obtain CDR1 andCDR3 Randomized gp120-Specific Fab Antibodies

The immunoglobulin gene phagemid expression vector, designated as pMT4to indicate the phagemid rather than the encoded Fab (MT4), contains theheavy and light chain sequences for expressing a Fab heterodimerantibody used as a template for the randomization of the complementaritydetermining regions (CDR) as shown herein. The pMT4 phagemid asdeposited expresses a soluble Fab antibody designated MT4 that binds tothe envelope glycoprotein of HIV-1, gp120. The selection of pMT4 fromscreening an IgG1K bone marrow library generated from an HIV-1seropositive individual (MT) and characterization thereof has beendescribed by Barbas et al., J. Mol. Biol., 230:812-823 (1993), thedisclosure of which is hereby incorporated by reference. The derivedamino acid residue sequences of both the heavy and light chain variabledomains of the Fab encoded by pMT4 has also been published by Barbas etal., J. Mol. Biol., 230:812-823 (1993).

The pMT4 plasmid was deposited with the American Type CultureCollection, 1301 Parklawn Drive, Rockville, Md., USA (ATCC). The depositof the plasmid-containing cells is listed under the name MT4 and hasbeen assigned the ATCC accession number 75574. This deposit was madeunder the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure and the Regulations thereunder (Budapest Treaty) as describedin the Examples.

Deposited pMT4 can be manipulated to insert the gene 3 membrane anchorto form pMT4-3 by derivation of nucleotide sequence encoding the anchorfrom pcomb3H-TT, described in Example 2B, by cleavage with, for example,Not I and Xho I, and moving the cleaved fragment into pMT4-3 at the sameNot I/Xho I sites.

The phagemid pMT4-3 was used in polymerase chain reaction (PCR)amplifications as described herein to introduce nucleotidesubstitutions, also referred to as nucleotide mutations, into both CDR1and CDR3 of the Fab heavy chain in the phagemid, to produce novel Fabantibodies that exhibit enhanced binding and neutralizingcharacteristics. The pMT4-3 vector was chosen as the template DNA onwhich randomization was performed as it was produced efficiently in E.coli and exhibited high affinity to gp120 and neutralization of HIV-1infection in cells.

The methods of producing higher affinity gp120 Fab antibodies of thisinvention that exhibit enhanced ability to inhibit HIV-1 infection ofcells involved the following steps presented as an overview: 1) Theheavy chain CDR1 of pMT4-3 was first randomized through the use ofpolymerase chain reaction (PCR); 2) Amplification products from the PCRcontaining randomized CDR1 were ligated back into pMT4-3 to form arandomized library; 3) Following expression of bacteriophage coatprotein 3-anchored Fab from the library, the Fab-expressing phage werepanned against gp120, strain IIIb, resulting in the selection ofgp120-specific CDR1 randomized Fabs; 4) The phagemid that encoded theselected Fabs were then subjected to randomization of 12 nucleotides inCDR3 in a one-step PCR amplification; 5) The resultant amplificationproducts having both CDR1 and a portion of CDR3 randomized were thenligated back into pMT4-3 and the expression and panning process wasrepeated; 6) Following selection of gp120-specific CDR1 and CDR3randomized Fabs, the corresponding phagemids were sequenced and theamino acid residue sequence was derived therefrom; and lastly 7) Bothsurface plasmon resonance analysis to determine the binding affinity andneutralization assays to determine the ability of the antibody toinhibit HIV-1 infection were performed as a final characterization ofthe resultant CDR1 and CDR3 randomized gp120-specific Fabs.

The above-enumerated steps are presented in detail below in thefollowing subsections.

B. Preparation of Randomized CDR1 of the Heavy Chain Variable Domain ofPhagemid MT4-3

1) Randomization by Overlap PCR

Libraries having a heavy chain variable domain randomized CDR1 wereproduced with two different approaches. Overlap PCR was used asdescribed herein for one approach. An alternative approach utilized aone-step PCR amplification process as described in Example 1B2) below.Randomization of heavy chain CDR1 in Fabs of this invention were derivedfrom the latter one-step PCR approach.

To randomize CDR1 in the heavy chain variable domain of pMT4-3 describedin Example 1A above, two separate PCR amplifications were performed asdescribed herein followed by a third overlap PCR amplification thatresulted in the annealing of the two previous amplification products,that was then followed by a third amplification. The nucleotide sequenceof the heavy chain variable domain of template pMT4-3 is shown in FIG. 4and is listed in SEQ ID NO 7. To facilitate subsequent PCRamplifications and subcloning, the pMT4-3 template was first mutagenizedby PCR to introduce a Hind III restriction site to cut at nucleotideposition 60 as shown in FIG. 4 and SEQ ID NO 7. The original guanine (G)nucleotide at position 60 in the coding strand was changed to an adenine(A) using PCR site-directed mutagenesis methods familiar to one ofordinary skill in the art. As a result of the site-directed mutagenesis,the pMT4-3 template contained an adenine nucleotide at position 60 alongwith the PCR amplified complementary thymine nucleotide. The codingsequence of the pMT4-3 heavy chain variable domain having themutagenized bases at position 60 is shown in FIG. 4 and SEQ ID NO 7.This mutagenized pMT4-3 was then used for all subsequent PCRamplifications to introduce CDR randomizations.

The nucleotide positions that were randomized in the heavy chain ofpMT4-3 began at nucleotide position 82 and ended at position 96. Thetemplate pMT4-3 DNA heavy chain sequence at that specified site encodedthe five total amino acid residues in CDR1. The amino acid residuesequence of CDR1 encoded by pMT4-3 was Asn-Phe-Val-Ile-His (SEQ ID NO8), as shown in the complete amino acid residue sequence of the heavychain variable domain in FIG. 1, labeled MT4. In FIG. 1 andcorresponding SEQ ID NO 1, the CDR1 of MT4-3 begins at amino acidresidue position 28 and ends at 32. This corresponds to the conservedKabat positions 31-35.

A pool of degenerate oligonucleotide primers, designated 12cdr1h3-fhaving the nucleotide formula shown below, synthesized by OperonTechnologies, Alameda, Calif., were used for randomizing the heavy chainCDR1 of pMT4-3. The five triplet codons for introducing randomizednucleotides had the repeating sequence NNS, where S can be either G or Cand N can be A, C, G or T.

The first PCR amplification resulted in the amplification of the regionof the heavy chain fragment in the pMT4-3 phagemid vector clone of mostof framework region 1 (FR1). To amplify this region, the followingprimer pairs were used. The 5′ oligonucleotide primer, FTX3, having thenucleotide sequence 5′-GCAATTAACCCTCACTAAAGGG-3′ (SEQ ID NO 9),hybridized to the noncoding strand of the heavy chain corresponding tothe region 5′ (vector sequence) of and including the first twonucleotides of FR1. The 3′ oligonucleotide primer, 12h3-b, having thenucleotide sequence 5′-AGAAGCTTGACAAGAAGAAACCTTC-3′ (SEQ ID NO 10)hybridized to the coding strand of the heavy chain ending at 15nucleotides from the end of framework 1. The oligonucleotide primerswere synthesized by Operon Technologies.

The PCR reaction was performed in a 100 microliter (ul) reactioncontaining one microgram (ug) of each of oligonucleotide primers FTX3and 12h3-b, 200 millimolar (mM) DNTP's (dATP, dCTP, dGTP, dTTP), 1.5 mMMgCl₂ Taq polymerase (5 units) (Perkin-Elmer Corp., Norwalk, Conn.), 10nanograms (ng) of template pMT4-3, and 10 ul of 10×PCR buffer purchasedcommercially (Perkin-Elmer Corp.). Thirty-five rounds of PCRamplification in a Perkin-Elmer Cetus 9600 GeneAmp PCR Systemthermocycler were then performed. The amplification cycle consisted ofdenaturing at 94 degrees C. (94 C.) for one minute, annealing at 50 C.for one minute, followed by extension at 72 C. for two minutes. Toobtain sufficient quantities of amplification product, 15 identical PCRreactions were performed.

The resultant PCR amplification products were then gel purified on a1.5% agarose gel using standard electroelution techniques as describedin “Molecular Cloning: A Laboratory Manual”, Sambrook et al., eds., ColdSpring Harbor, N.Y. (1989). Briefly, after gel electrophoresis of thedigested PCR amplified Fab-display encoding synthetic binding sites, theregion of the gel containing the DNA fragments of predetermined size wasexcised, electroeluted into a dialysis membrane, ethanol precipitatedand resuspended in buffer containing 10 millimolar (mM) Tris-HCl[Tris(hydroxymethyl)aminomethane-hydrochloride] at pH 7.5 and 1 mM EDTA(ethylenediaminetetraacetic acid) to a final concentration of 50nanograms/milliliter (ng/ml).

The purified resultant PCR amplification products from the firstreaction were then used in an overlap extension PCR reaction with theproducts of the second PCR reaction, both as described below, torecombine the two products into reconstructed heavy chains containingrandomized CDR1.

The second PCR reaction resulted in the amplification of the heavy chainoverlapping framework 1 with the above products and extending 3′ offramework 4 of the heavy chain. To amplify this region for randomizingthe encoded five amino acid residue sequence of CDR1, the followingprimer pairs were used. The 5′ coding oligonucleotide primer pool asdescribed above, designated 12cdr1h3-f, had the nucleotide sequencerepresented by the formula,5′-GAAGGTTTCTTGTCAAGCTTCTGGATACAGATTCAGTNNSNNSNNSNNSNNSTGGGTGCGCCAGGCCCCC-3′ (SEQ ID NO 11), where N can be either A, C, G, or Tand S is G or C. The 3′ noncoding primer, R3B, hybridized to the codingstrand at the 3′ end of CH1 having the sequence5′-TTGATATTCACAAACGAATGG-3′ (SEQ ID NO 12). The 5′ end of theoligonucleotide primer pool is complementary to the 3′ end of framework1 and the 3′ end of the oligonucleotide primer pool is complementary tothe 5′ end of framework 2. The region between the two specified ends ofthe primer pool is represented by a 15-mer NNS degeneracy. The secondPCR reaction was performed on a second aliquot of pMT4-3 template in a100 ul reaction as described above containing 1 ug of each ofoligonucleotide primers as described. The resultant PCR products encodeda diverse population of randomized heavy chain CDR1 regions of 5 aminoacid residues in length. The products were then gel purified asdescribed above.

For the annealing reaction of the two PCR amplifications, 1 mg each ofgel purified products from the first and second PCR reactions were thenadmixed and fused in the absence of primers for 35 cycles of PCR asdescribed above. The resultant fusion product was then amplified with 1ug each of FTX3 and R3B oligonucleotide primers as a primer pair in afinal PCR reaction to form a complete heavy chain fragment by overlapextension. The overlap PCR amplification was performed as describedabove.

To obtain sufficient quantities of amplification product, 15 identicaloverlap PCR reactions were performed. The resulting heavy chainfragments extended from 5′ to framework 1 to the end of CH1 and hadrandomized CDR1 for encoding 5 amino acid residues. The CDR1-randomizedheavy chain fragment amplification products of approximately 880 basepairs (bp) in length in each of the 15 reactions were first pooled andthen gel purified as described above prior to their religation into thepMT4-3 surface display phagemid expression vector to form a library forsubsequent screening against gp120. The ligation procedure in creatingexpression vector libraries and the subsequent expression of the heavychain CDR1-randomized pMT4-3 clones was performed as described inExample 1C.

2) Randomization by One-Step PCR

An alternative approach for randomizing CDR1 in a heavy chain variabledomain to produce heavy chain CDR1-randomized Fabs of this invention wasperformed with one PCR mutagenesis step as described herein. The Fabs ofthis invention having a CDR1-randomized heavy chain variable domain asdescribed in the following Examples were obtained from screening thephagemid-displayed Fab libraries produced from the one-step PCRapproach.

Instead of performing three PCR amplifications to produce onefull-length variable domain having a mutagenized CDR1 as described inExample 1B1) for overlap PCR, in one-step PCR, the amplifications wereperformed to utilize the Hind III restriction site preceding the heavychain CDR1 that was previously introduced into the pMT4-3 phagemidvector template as described in Example 1B1). The nucleotide sequence ofthe heavy chain variable domain of template pMT4-3 is shown in FIG. 4and is listed in SEQ ID NO 7. The region of the template that wasrandomized for introducing mutations into CDR1 was as previouslydescribed for overlap PCR. Thus, to randomize CDR1, the previouslydescribed primers, the mutagenizing coding primer 12cdr1h3-f (SEQ ID NO11) and the 3′ noncoding primer R3B (SEQ ID NO 12), were used tointroduce randomized nucleotides into the heavy chain CDR1 with the PCRprotocol as described in Example 1B1) and to amplify sequences beginningat nucleotide position 45 as shown in FIG. 4 and in SEQ ID NO 7extending into CH1 as previously described.

The resultant PCR products were gel purified, digested with Hind III andSpe I, and gel purified. The Hind III/Spe I digest resulted in a PCRproduct having a Hind III 5′ coding overhanging end, the 5′ end of whichcorresponded with nucleotide position 60 of the pMT4-3 heavy chainvariable domain sequence shown in FIG. 4 and in SEQ ID NO 7 and a Spe I3′ end in framework 4 of the heavy chain variable domain.

The double digested PCR products were then directionally ligated into asimilarly digested pMT4-3 vector in which the unmutagenized light chainvariable domain for encoding Fab MT4 was retained. The Hind III digestof pMT4-3, due to the introduced Hind III site as described in Example1B1), cut between nucleotide positions 59 and 60 as shown in FIG. 4 ofpMT4-3 and in SEQ ID NO 7 leaving the adenine base in position 59 at the3′ coding end of a linearized vector along with a noncoding overhangingend to allow for directional ligation of the digested PCR products intothe digested pMT4-3 vector.

Thus, the library of randomized products that were double digested withHind III and Spe I were directionally ligated into a similarly digestedpMT4-3 vector to form a library of circularized pMT4-3 vectors having aCDR1-randomized heavy chain variable domain and a unmutagenized lightchain variable domain from the pMT4-3 vector as described in Example 1C.The ligation resulted in the in-frame ligation of the PCR amplifiedrandomized heavy chain variable domain beginning at position 60 with thenucleotide sequence encoding the 5′ end of the heavy chain fromnucleotide positions 1 to 59. The resultant phagemid library producedfrom one-step PCR for introducing randomized nucleotide sequences intothe heavy chain variable domain of pMT4-3 were then screened asdescribed in Example 1D and used to derived the CDR1-randomized heavychain variable domain Fabs described in this invention.

C. Preparation of a Phagemid-Displayed Fabs Having Randomized CDR1

The phagemid pMT4-3 containing heavy and light chain variable domainsequences is a pComb3 phagemid expression vector that provides for theexpression of phage-displayed anchored proteins. The pComb3 expressionvector has been designed to allow for anchoring of expressed proteins onthe bacteriophage coat protein 3. Gene III of filamentous phage encodesthis 406-residue minor phage coat protein, cpIII (cp3), which isexpressed prior to extrusion in the phage assembly process on abacterial membrane and accumulates on the inner membrane facing into theperiplasm of E. coli.

In practicing this invention to obtain expression of Fab-displayedproteins containing a randomized CDR on the phage surface, the heavy (Fdconsisting of V_(H) and C_(H)1) and light (kappa) chains (V_(L), C_(L))of antibodies were first targeted to the periplasm of E. coli for theassembly of heterodimeric Fab molecules.

In this system, the first cistron encoded a periplasmic secretion signal(pelB leader) operatively linked to the fusion protein, Fd-cpIII. Thesecond cistron encoded a second pelB leader operatively linked to akappa light chain. The presence of the pelB leader facilitated thecoordinated but separate secretion of both the fusion protein containingthe synthetic binding site and light chain from the bacterial cytoplasminto the periplasmic space.

In this process, each chain was delivered to the periplasmic space bythe pelB leader sequence, which was subsequently cleaved. The heavychain containing the synthetic binding was anchored in the membrane bythe cpIII membrane anchor domain while the light chain was secreted intothe periplasm. Fab molecules were formed from the binding of the heavychain with the soluble light chains.

The phagemid vector, designated pComb3, allowed for both surface displayand soluble forms of Fabs. The vector was designed for the cloning ofcombinatorial Fab libraries. Xho I and Spe I sites were provided forcloning complete PCR-amplified heavy chain (Fd) sequences consisting ofthe region beginning with framework 1 and extending through framework 4.A Hind III site engineered into pMT4-3 provided for directional in-frameligation of partial PCR amplified heavy chain variable domain fragments.An Aat II restriction site is also present in the heavy chain CDR3. Thepresence of the Aat II site allowed for the insertion of Xho I/Aat IIdigests of the PCR products prepared in Example 1E that containsequences beginning with framework 1 and extending to the end of theCDR3 domain in which the sequences for encoding both mutagenized CDR1and CDR3 are located. The insertion of an Xho I/Aat II digest intopMT4-3 as described in Example 1F resulted in the fusion of therandomized pMT4-3 heavy chain variable domain with framework 4 alreadypresent in the pMT4-3 vector. Thus, the ligation of the final heavychain mutagenized nucleotide sequence prepared in Example 1E resulted inthe in-frame ligation of a complete heavy chain fragment consisting ofPCR amplified framework 1 through CDR3 and retained pMT4-3 framework 4.The cloning sites in the pComb3 expression vectors were compatible withpreviously reported mouse and human PCR primers as described by Huse etal., Science, 246:1275-1281 (1989) and Persson et al., Proc. Natl. Acad.Sci., USA, 88:2432-2436 (1991). The nucleotide sequence of the pelB, aleader sequence for directing the expressed protein to the periplasmicspace, was as reported by Huse et al., Science, 246:1275-1281 (1989).

The vector also contained a ribosome binding site as described by Shineet al., Nature, 254:34 (1975). The sequence of the phagemid vector,pBluescript, which includes ColE1 and F1 origins and a beta-lactamasegene, has been previously described by Short et al., Nuc. Acids Res.,16:7583-7600 (1988) and has the GenBank Accession Number 52330 for thecomplete sequence. Additional restriction sites, Sal I, Acc I, Hinc II,Cla I, Hind III, Eco RV, Pst I and Sma I, located between the Xho I andSpe I sites of the empty vector were derived from a 51 base pair stufferfragment of pBluescript as described by Short et al., Nuc. Acids Res.,16:7583-7600 (1988). A nucleotide sequence that encodes a flexible 5amino acid residue tether sequence which lacks an ordered secondarystructure was juxtaposed between the Fab and cp3 nucleotide domains sothat interaction in the expressed fusion protein was minimized.

Thus, the resultant combinatorial vector, pComb3, consisted of a DNAmolecule having two cassettes to express one fusion protein, Fd/cp3, andone soluble protein, the light chain. The vector also containednucleotide residue sequences for the following operatively linkedelements listed in a 5′ to 3′ direction: a first cassette consisting ofLacZ promoter/operator sequences; a Not I restriction site; a ribosomebinding site; a pelB leader; a spacer region; a cloning region borderedby 5′ Xho and 3′ Spe I restriction sites; the tether sequence; thesequences encoding bacteriophage cp3 followed by a stop codon; a Nhe Irestriction site located between the two cassettes; a second lacZpromoter/operator sequence followed by an expression control ribosomebinding site; a pelB leader; a spacer region; a cloning region borderedby 5′ Sac I and a 3′ Xba I restriction sites followed by expressioncontrol stop sequences and a second Not I restriction site.

In the above expression vector, the Fd/cp3 fusion and light chainproteins were placed under the control of separate lac promoter/operatorsequences and directed to the periplasmic space by pelB leader sequencesfor functional assembly on the membrane. Inclusion of the phage F1intergenic region in the vector allowed for the packaging ofsingle-stranded phagemid with the aid of helper phage. The use of helperphage superinfection allowed for the expression of two forms of cp3.Consequently, normal phage morphogenesis was perturbed by competitionbetween the Fd/cp3 fusion and the native cp3 of the helper phage forincorporation into the virion. The resulting packaged phagemid carriednative cp3, which is necessary for infection, and the encoded Fab fusionprotein, which is displayed for selection. Fusion with the C-terminaldomain was necessitated by the phagemid approach because fusion with theinfective N-terminal domain would render the host cell resistant toinfection.

The pComb3 expression vector described above forms the basic constructof the MT4 Fab display phagemid expression vector, also referred to aspMT4-3, used in this invention for the production of synthetic human Fabantibodies against gp120 of HIV-1.

1) Phagemid Library Construction

In order to obtain expressed synthetic human Fab antibodies having bothheavy and light chain variable domains, phagemid libraries wereconstructed. The libraries provided for the expression of recombinanthuman Fab antibodies having heavy and light chains where CDR1 wasrandomized in the heavy chain as described in Example 1B.

For preparation of phagemid libraries for expressing the PCR productsprepared in Example 1B, the PCR products from overlap PCR were firstdigested with Xho I and Spe I and separately ligated with similarlydigested original (i.e., not randomized) pMT4-3 phagemid expressionvectors prepared as described in Example 1A. The Xho I and Spe I siteswere present in the framework 1 region and CH1 domain, respectively. Theligation resulted in operatively linking the heavy chain variable domainfrom framework 1 to the end of framework 4 to the pMT4-3 vector, located5′ to the cp3 gene. Since the amplification products were inserted intothe template pMT4-3 expression vector that originally had both heavy andlight chain variable domain sequences for expressing Fab MT4, only theheavy chain domain was replaced leaving the rest of the pMT4-3expression vector unchanged. In other words, the newly randomized CDR1heavy chain amplification products were religated back into pMT4-3 withthe original pMT4-3 light chain variable domain. Thus, upon expressionfrom the recombinant clones, the expressed Fabs contained aCDR1-randomized heavy chain and the pMT4-3 light chain sequence, thelatter of which is shown in FIG. 2 and in SEQ ID NO 6. The PCR productsfrom the one-step PCR approach were digested with Hind III and Spe I andthen separately ligated with similarly digested pMT4-3 expressionvectors. The result was the same as that described for overlap PCR.

The pMT4-3 light chain variable domain nucleotide sequence was retainedunchanged throughout the mutagenesis procedure. Therefore, all thepreferred anti-gp120 Fab antibodies obtained by the methods of thisinvention as described in Example 1 contain the light chain amino acidresidue sequence encoded by the original pMT4-3.

Phagemid libraries for expressing each of the Fab display syntheticbinding sites of this invention were prepared in the followingprocedure. To form circularized vectors containing the PCR productinsert, 640 ng of the digested PCR products were admixed with 2 ug ofthe linearized pMT4-3 phagemid vector and ligation was allowed toproceed overnight at room temperature using 10 units of BRL ligase(Gaithersburg, Md.) in BRL ligase buffer in a reaction volume of 150 ul.Five separate ligation reactions were performed to increase the size ofthe phage library having randomized CDR1. Following the ligationreactions, the circularized DNA was precipitated at −20 degrees Celsius(−20 C.) for 2 hours by the admixture of 2 ul of 20 mg/ml glycogen, 15ul of 3 M sodium acetate at pH 5.2 and 300 ul of ethanol. DNA was thenpelleted by microcentrifugation at 4 C. for 15 minutes. The DNA pelletwas washed with cold 70% ethanol and dried under vacuum. The pellet wasresuspended in 10 ul of water and transformed by electroporation into300 ul of E. coli XL1-Blue cells to form a phage library.

After transformation, to isolate phage on which Fabs having mutagenizedCDR1 were induced for subsequent panning on the gp120 glycoprotein asdescribed in Example 1D, 3 ml of SOC medium (SOC was prepared byadmixture of 20 grams (g) bacto-tryptone, 5 g yeast extract and 0.5 gNaCl in 1 liter of water, adjusting the pH to 7.5, autoclaving followedby admixture of 20 mM glucose) were admixed and the culture was shakenat 220 rpm for 1 hour at 37 C. After that, 10 ml of SB (SB was preparedby admixing 30 g tryptone, 20 g yeast extract, and 10 g Mops buffer perliter with pH adjusted to 7) containing 20 ug/ml carbenicillin and 10ug/ml tetracycline were admixed and the admixture was shaken at 300 rpmfor an additional hour. This resultant admixture was admixed to 100 mlSB containing 50 ug/ml carbenicillin and 10 ug/ml tetracycline andshaken for 1 hour, after which helper phage VCSM13 (10¹² pfu) wereadmixed and the admixture was shaken for an additional 2 hours at 37 C.After this time, 70 ug/ml kanamycin was admixed and maintained at 30 C.overnight. The lower temperature resulted in better heterodimerincorporation on the surface of the phage. The supernatant was clearedby centrifugation (4000 rpm for 15 minutes in a JA10 rotor at 4 C.).Phage were precipitated by admixture of 4% (w/v) polyethylene glycol8000 and 3% (w/v) NaCl and maintained on ice for 30 minutes, followed bycentrifugation (9000 rpm for 20 minutes in a JA10 rotor at 4 C.). Phagepellets were resuspended in 2 ml of PBS and microcentrifuged for threeminutes to pellet debris, transferred to fresh tubes and stored at −20C. for subsequent screening as described below.

For determining the titering colony forming units (cfu), phage (packagedphagemid) were diluted in SB and 1 ul was used to infect 50 ul of fresh(A_(OD600)=1) E. coli XL1-Blue cells grown in SB containing 10 ug/mltetracycline. Phage and cells were maintained at room temperature for 15minutes and then directly plated on LB/carbenicillin plates. Theresulting phage library containing randomized CDR1 heavy chain geneswere thus found to contain about 2×10⁷ phage particles (cfu).

For subsequent screening of the library, the library was amplified to apopulation size of about 10¹¹ cfu containing a diversity of 2×10⁷particles, and the amplified library used as needed. Amplification of aphage library was conducted as described below for amplifying elutedphage in Example 1D1).

D. Selection of Anti-gp120 Fab Heterodimers Expressed on Phage Surfaces

1) Multiple Pannings of the Phage Library

The phage library produced in Example 1C from the one-step PCR approachwas panned against recombinant gp120 of HIV-1 strain IIIb as describedherein on coated microtiter plate select for anti-HIV-1 heterodimers.

The panning procedure used, comprised of several rounds of recognitionand replication, was a modification of that originally described byParmley and Smith (Parmley et al., Gene, 73:305-318 (1988). Four roundsof panning were performed to enrich for specific antigen-binding clones.For this procedure, four wells of a microtiter plate (Costar 3690) werecoated overnight at 4 C. with 25 ul of 40 ug/ml gp120 (AmericanBiotechnologies, Ossining, N.Y.) prepared above in 0.1 M bicarbonate, pH8.6. The wells were washed twice with water and blocked by completelyfilling the well with 3% (w/v) BSA in PBS and maintaining the plate at37 C. for one hour. After the blocking solution was shaken out, 50 ul ofthe amplified phage suspension prepared above (typically 10¹¹ cfu) wereadmixed to each well, and the plate was maintained for 2 hours at 37 C.

Phage were removed and the plate was washed once with water. Each wellwas then washed 10 times with TBS/Tween (50 mM Tris-HCl at pH 7.5, 150mM NaCl, 0.5% Tween 20) over a period of 1 hour at room temperaturewhere the washing consisted of pipetting up and down to wash the well,each time allowing the well to remain completely filled with TBS/Tweenbetween washings. The plate was washed once more with distilled waterand adherent phage were eluted by the addition of 50 ul of elutionbuffer (0.1 M HCl, adjusted to pH 2.2 with solid glycine, containing 1mg/ml BSA) to each well followed by maintenance at room temperature for10 minutes. The elution buffer was pipetted up and down several times,removed, and neutralized with 3 ul of 2 M Tris base per 50 ul of elutionbuffer used.

The population of eluted phage was amplified to increase the totalnumber of particles in the library and to facilitate subsequent titeringof the library. To that end, eluted phage were used to infect 2 ml offresh (OD₆₀₀=1) E. coli XL1-Blue cells for 15 minutes at roomtemperature, after which time 10 ml of SE containing 20 ug/mlcarbenicillin and 10 ug/ml tetracycline were admixed. Aliquots of 20,10, and 1/10 ul were removed from the culture for plating to determinethe number of phage (packaged phagemids) that were eluted from theplate. The culture was shaken for 1 hour at 37 C., after which it wasadded to 100 ml of SB containing 50 ug/ml carbenicillin and 10 ug/mltetracycline and shaken for 1 hour. Helper phage VCSM13 (10¹² pfu) werethen added and the culture was shaken for an additional 2 hours. Afterthis time, 70 ug/ml kanamycin were added and the culture was incubatedat 37 C. overnight to form a population of amplified phage. Phagepreparation and further panning were repeated as described above for atotal of 4 rounds of panning.

Following each round of panning, the percentage yield of phage can bedetermined, where % yield—(number of phage eluted/number of phageapplied)×100. The initial phage input ratio was calculated based ontitering the amplified phage population on selective plates to beapproximately 10¹¹ cfu for each round of panning. The final phage outputratio can be determined by infecting two ml of logarithmic phaseXL1-Blue cells as described above and plating aliquots on selectiveplates. Typically, the output of phage eluted from the panning procedurewas about 10⁵-10⁶ cfu. From this panning procedure, clones were selectedfrom the Fab library for their ability to bind to glycosylatedrecombinant gp120 from the IIIB strain of HIV-1. The selected clones hadrandomized CDR1 heavy chain variable domains and the light chainvariable domain sequence from pMT4-3.

The resulting selected clones that bound gp120 were sequenced todetermine the CDR1 heavy chain sequence as described in Example 1D1).

2) Nucleic Acid Sequence Analysis Comparison Between HIV-1 SpecificMonoclonal Antibody Fabs and the Corresponding Derived Amino AcidResidue Sequence Following Randomization of CDR1

Nucleic acid sequencing of the CDR1 randomized clones produced inExample 1D1) was performed on 12 randomly chosen soluble double-strandedFab-expressing DNA using Sequenase 1.0 (USB, Cleveland, Ohio). Alignmentof derived sequences with one another and with the Genbank database madeuse of the MacVector suite of programs. The derived heavy chain aminoacid residue sequences of 12 selected specific synthetic gp120-specificFabs and MT4 are shown in FIG. 5 under column heading Experiment A. Thealignment of the variable heavy chain domains as shown in FIG. 5 revealsthat in CDR1 the original MT4 gp120-specific Fab obtained from screeninga bone marrow library from an HIV-1 seropositive individual, the aminoacid residue sequence was Asn-Phe-Val-Ile-His (SEQ ID NO 8). Sequencecomparisons indicated a preference for asparagine (N) at position 31, anaromatic residue at position 32, serine (S) or threonine (T) primarilyat position 33, branched hydrophobic residues at position 34, andhydrophobic and/or aromatic residues at position 35. The amino acidresidue positions 31-35 have been referred to on the basis on conservedKabat position numbering. The actual amino acid residue position in thecomplete variable domain amino acid residue sequences shown in FIG. 1begin and end at 28-32, respectively, resulting from a shortenedframework 1. The 12 amino acid residue sequences, indicated inExperiment A for CDR1 of the 12 selected Fabs have been assigned SEQ IDNOs 14-25 as follows: Arg-Tyr-Thr-Val-Phe (SEQ ID NO 14),Asn-Trp-Ser-Val-Met (SEQ ID NO 15), Gly-Tyr-Thr-Leu-Met (SEQ ID NO 16),Asn-Phe-Thr-Leu-Leu (SEQ ID NO 17), His-Tyr-Ser-Leu-Met (SEQ ID NO 18),Asn-Trp-Val-Val-His (SEQ ID NO 19), Asn-Phe-Ser-Ile-Met (SEQ ID NO 20),Asn-Phe-Ala-Ile-His (SEQ ID NO 21), Asn-Phe-Thr-Met-Val (SEQ ID NO 22),Asn-Phe-Thr-Leu-Gln (SEQ ID NO 23), Tyr-Phe-Thr-Met-His (SEQ ID NO 24),and Ser-Tyr-Pro-Leu-His (SEQ ID NO 25).

The CDR3 in the heavy chain domain of the selected clones was thenrandomized as described in Example 1E to form a phagemid havingrandomized CDR1 and CDR3 for selection on gp120.

E. Preparation of Randomized CDR3 of the Heavy Chain Variable Domain ofCDR1-Randomized Fab-Expressing Clones

The selected CDR1-randomized heavy chain Fab-expressing clones fromExample 1D were subjected to additional PCR amplifications in order toselectively randomize 12 nucleotides in the heavy chain CDR3 sequence ofthose clones. The nucleotide position in which the nucleotiderandomization was directed began at nucleotide position 292 and ended atposition 303 as shown in FIG. 4 and SEQ ID NO 7, of the heavy chainsequence of the original pMT4-3 phagemid. The 12 nucleotides asspecified were randomized with a pool of degenerate oligonucleotideprimers have a repeat of NNK, where N can either be A, C, G or T, and Kis either G or T, as written in the 5′ to 3′ direction of the codingstrand. In this instance, since the 3′ end of the heavy chain was beingrandomized, the oligonucleotide primer pool was the 3′ primer. Thedegenerate oligonucleotides, designed for incorporating the randomizednucleotides, thus was the anti-sense or noncoding strand that hybridizedto the coding strand in the PCR amplification. The complementarysequence of NNK is NNM, where N has been defined above, and M is eitherA or C, and NNM is written in the 3′ to 5′ direction. The NNM repeat iswritten MNN in the convention of 5′ to 3′. The degeneracy repeats for 4times in the degenerate oligonucleotide pool.

The noncoding degenerate oligonucleotide primer pool, designated12-4-cdr3, written in the 5′ to 3′ direction, had the nucleotidesequence 5′-CCCTTTGCCCCAGACGTCCATATAATAATTGTCCTGGGGAGAATCATCMNNMNNMNNMNNCCCCACTCTCGCACA-3′ (SEQ ID NO 13). The 12-4-cdr3 primer had anatural Aat II restriction site for allowing the insertion of an Xho Iand Aat II restricted amplification product into a similarly digestedpMT4-3 phagemid as described below in Example 1F. The 5′ oligonucleotideprimer used in amplifying the CDR1-mutagenized selected clones fromExample 1D was FTX3 described in Example 1C having the nucleotidesequence in SEQ ID NO 9.

The PCR amplification was performed as described in Example 1C with theexception that overlap PCR was not necessitated in this case as theentire heavy chain variable domain was amplified in one reactionextending from before and including framework 1 to the middle offramework 4. The resultant PCR products were purified as described inExample 1C and subsequently reinserted as described in Example 1F into alinearized pMT4-3 phagemid having the light chain variable domain ofpMT4-3.

F. Preparation of a Phagemid-Displayed Fabs Having Randomized CDR1 andCDR3

The amplification products produced as described in Example 1E in whichthe heavy chain variable domain CDR1 and a 12 nucleotide sequence ofCDR3 were randomized were then digested with Xho I and Aat II forligation in a similarly digested original pMT4-3 phagemid. The Xho Isite was present in the first six nucleotides of the heavy chainframework 1 region while the Aat II site was the last six nucleotides ofCDR3. The ligation of a population of amplified Xho I/Aat II restrictiondigested heavy chain CDR1- and CDR3-mutagenized products resulted in thein-frame ligation of this portion of the heavy chain to the framework 4region retained from pMT4-3. Thus, in step-wise randomizations of CDR1and CDR3, only those specified nucleotides were randomized in thenucleotide sequence of pMT4-3 heavy chain variable domain. As previouslydescribed, the light chain of pMT4-3 was retained unchanged.

The resultant ligated pMT4-3 having a randomized heavy chain domain inthe CDR1 and CDR3 was then processed as described in Example 1C forsubsequent transformation into XL1-Blue. Following transformation, thephage were expressed as previously described to result in thephage-display of Fab antibodies having a randomized heavy chain domainin the CDR1 and CDR3 on a library of phage. The phage library wastitered as before and contained about 8×10⁶ phage particles (cfu). Forsubsequent manipulations, the library was first amplified to provide astock library of 10¹¹ cfu as described in Example 1D.

A second panning selection protocol which included 6 rounds of panningwas then performed on the amplified library as described in Example 1Dto obtain Fabs that bound to gp120. From the panning procedure, eightgp120-specific Fab heterodimers having heavy chain CDR1 and CDR3randomized amino acid residue sequences and the original MT4 light chainsequence shown in FIG. 2 and listed in SEQ ID NO 6 were selected andfurther characterized as described in Examples 1G and 1H.

G. Nucleic Acid Sequence Analysis Comparison Between HIV-1 SpecificMonoclonal Antibody Fabs and the Corresponding Derived Amino AcidResidue Sequence

In order to further characterize, by sequence analysis and functionalcharacteristics, the specificity of the mutagenized heterodimersexpressed on the surface of phage as described above, soluble Fabheterodimers from acid eluted phage were prepared.

To prepare soluble heterodimers, phagemid DNA from the gp120-reactiveclones prepared above was isolated and digested with Spe I and Nhe I.Digestion with these restriction enzymes produced compatible cohesiveends. The 4.7-kb DNA fragment lacking the gene III portion wasgel-purified (0.6% agarose) and self-ligated. Transformation of E. coliXL1-Blue afforded the isolation of recombinants lacking the cpIIIfragment. Clones were examined for removal of the cpIII fragment by XhoI—Xba I digestion, which should yield an 1.6-kb fragment. Clones weregrown in 100 ml SB containing 50 ug/ml carbenicillin and 20 mM MgCl₂ at37 C. until an OD₆₀₀ of 0.2 was achieved. IPTG (1 mM) was added and theculture grown overnight at 30 C. (growth at 37 C. provides only a slightreduction in heterodimer yield). Cells were pelleted by centrifugationat 4000 rpm for 15 minutes in a JA10 rotor at 4 C. Cells wereresuspended in 4 ml PBS containing 34 ug/ml phenylmethylsulfonylfluoride (PMSF) and lysed by sonication on ice (2-4 minutes at 50%duty). Debris was pelleted by centrifugation at 14,000 rpm in a JA20rotor at 4 C. for 15 minutes. The supernatant was stored at −20 C. Forthe study of a large number of clones, 10 ml cultures providedsufficient heterodimer for analysis. In this case, sonications wereperformed in 2 ml of buffer.

Nucleic acid sequencing was performed on the soluble double-strandedFab-expressing DNA using Sequenase 1.0 (USB, Cleveland, Ohio). Thederived heavy chain amino acid residue sequences of four selectedspecific synthetic gp120-specific Fabs are shown in FIG. 1. The selectedsynthetic Fabs have been designated 3b1, 3b3, 3b4 and 3b9. The derivedheavy chain amino acid residue sequences of eight selected specificsynthetic gp120-specific Fabs are shown in FIG. 5. The eight sequencesshown in FIG. 5 include Kabat identified amino acid residues 31 to 35 ofCDR1 and amino acid residues 96 to 99 of CDR3 of the Fabs 3b1, 3b3, 3b4,and 3b9 which are also shown in FIG. 1. Kabat positions 96 to 99correspond to actual amino acid residue positions 98-101 as shown inFIG. 1 and in the sequence listing (SEQ ID NOs 1-5). The selectedsynthetic Fabs have been designated 3b1, 3b2, 3b3, 3b4, 3b6, 3b7, 3b8,and 3b9.

The amino acid residues indicated in Experiment B in FIG. 5 for aminoacid residues 31 to 35 of CDR1 have been assigned SEQ ID NOs 26-33 asfollows: Asn-Phe-Thr-Leu-Met (3b1, SEQ ID NO 26), Asn-Tyr-Thr-Ile-Met(3b2, SEQ ID NO 27), Asn-Phe-Thr-Val-His (3b3, SEQ ID NO 28),Asn-Tyr-Thr-Leu-Ile (3b4, SEQ ID NO 29), Asn-Phe-Ile-Ile-Met (3b6, SEQID NO 30), Asn-Phe-Ser-Ile-Met (3b7, SEQ ID NO 31), Asn-Tyr-Thr-Ile-Gln(3b8, SEQ ID NO 32) and Asn-Phe-Thr-Val-His (3b9, SEQ ID NO 33). Theamino acid residues indicated in Experiment B in FIG. 5 for Kabatidentified amino acid residues 96 to 99 of CDR3 have been assigned SEQID NOs 34-42 as follows: Pro-Tyr-Ser-Trp (MT4, SEQ ID NO 34),Gln-Trp-Asn-Trp (3b1, SEQ ID NO 35), Pro-Trp-Thr-Trp (3b2, SEQ ID NO36), Glu-Trp-Gly-Trp (3b3, SEQ ID NO 37), Pro-Trp-Asn-Trp (3b4, SEQ IDNO 38), Leu-Trp-Asn-Trp (3b6, SEQ ID NO 39), Ser-Trp-Arg-Trp (3b7, SEQID NO 40), Pro-Tyr-Ser-Trp (3b8, SEQ ID NO 41), and Pro-Trp-Arg-Trp(3b9, SEQ ID NO 42).

Alignment of derived sequences with one another and with the Genbankdatabase made use of the MacVector suite of programs. For analysis ofheavy chain CDR3 sequences as described by Sanz, J. Immunol.,147:1720-1729 (1991), the most 5′ nucleotide was considered to be thefirst nucleotide after codon 95 of the H chain variable region accordingto Kabat et al, Sequences of Proteins of Immunological Interest, USDept. of Health and Human Services, Washington, DC (1991).

In assessing the randomization of amino acid residue sequence in CDR3following 6 rounds of selection for binding to IIIb-derived gp120,conservation of particular amino acid residues is also noted.Specifically, the amino acid residues randomized in MT4 Fab werePro-Tyr-Ser-Trp as shown in FIG. 1 from the third to the sixth aminoacid residue positions in MT4. This corresponds to amino acid residueposition 98-101 in SEQ ID NO 1 (Kabat positions 96-99). The tryptophanresidue remained unchanged throughout the randomization and selectionprocedures. In all four preferred CDR1-and CDR3-randomized Fabs, thetyrosine amino acid residue was replaced by a tryptophan, thereinexhibiting selection pressures for the particular amino acid residue inthis position. The derived amino acid residue sequence of 3b8 isidentical to that of MT4 and may indicate some contamination in theCDR1- and CDR3-randomized library.

In order to assess what effect the randomized amino acid residuesequences in the heavy chain CDR1 and CDR3 had on the functionalabilities of the soluble Fabs, both binding affinity studies andneutralization assays were performed as described in Example 1H. Fourclones, 3b1, 3b3, 3b4, and 3b9, were chosen for further study. Thesefour clones have a sequence relatedness to one another characterized bysmall changes in amino acid sequence and also display the most dramaticchange in amino acid residue identity at positions 96 and 98.

H. Functional Characterization of gp120-Specific Fabs Having RandomizedCDR1 and CDR3 Heavy Chain Domains

1) Binding Affinity Analysis

The four selected Fabs, 3b1, 3b3, 3b4 and 3b9, having randomized aminoacid residue sequences in the entire CDR1 and in four of the 18 aminoacid residues of the CDR3, were used in binding affinity assays. Surfaceplasmon resonance assays were performed in a BIAcore binding affinitymeasurement apparatus (Pharmacia, Piscataway, N.J.) followingmanufacturer's instructions to determine whether the affinity of therandomized Fabs had improved binding affinities as compared to theoriginal MT4 Fab from which the new Fabs were derived.

In the assay, the gp120 glycoprotein, isolated from both MN (AgMed,Cambridge, Mass.) and IIIb (American Bio-Technologies) strains of HIV-1were coated onto gold chips. The four soluble Fabs listed above,including MT4, were then separately admixed with the gp120-coated goldchips and the binding of the Fabs to the ligand was measured in theBIAcore apparatus. As the mass of the gold chips increases due to thebinding of the Fabs to the ligand, the refractive index of the chipsincreases indicating a coordinate increase in the increase of the mass.Measurements of the “on” rate are made in addition to the “off” rates,the latter of which occurs as the Fabs begin to dissociate from theligand and the mass coordinately decreases, thereby allowing ameasurement of the change of the refractive index. Briefly, the sensorchip was activated for immobilization with N-hydroxysuccinimide anN-ethyl-N′-(3-diethyl aminopropyl) carbodiimide. The proteins,MN-derived or IIIb-derived gp120, were coupled to the surface byinjection of 50 ul of a 50 ug/ml sample. Excess activated esters werequenched with 15 ul ethanolamine, 1 M pH 8.5. Typically, 4000 resonanceunits were immobilized. Binding of Fab fragments to immobilized gp120was studied by injection of Fab in a range of concentrations (0.5 to 10ug/ml) at a flow rate of 5 ul/minute. The association was monitored asthe increase in resonance units per unit time. Dissociation measurementswere acquired following the end of the association phase but with a flowrate of 50 ul/minute. The binding surface was regenerated with HCl, 1MNaCl, pH 3 and remained active for 20-40 measurements. The associationand dissociation rate constants, k_(on) and k_(off), were determinedfrom a series of measurements as described in Barbas et al., Gene, inpress, (1993); Altschuh et al., Biochemistry, 31:6298-6304 (1992); andKarlsson et al., J. Immunol. Methods, 145:229-240, (1991). Equilibriumassociation and dissociation constants were deduced from the rateconstants.

Both the on and off measurements for all four randomized Fabs and MT4were collected. From these measurements, the association constant,K_(a), was determined by dividing the on constant (K_(on) (M⁻¹s⁻¹) withthe off (K_(off) (s⁻¹) constant. The dissociation constant, K_(d), canalso be determined from these measurements and is expressed asK_(off)/K_(on). The measurement for both the on and off values for gp120isolated from both MN and IIIb strains are shown, respectively, inTables 1 and 2 below. In addition, the calculated value of the K_(a) andK_(d) from the measurement of the binding affinity is shown for all theFabs analyzed.

TABLE 1 (MN Strain) Fab K_(on) (^(M-1S-1)) K_(off)(S⁻¹) K_(a)(M⁻¹)K_(d)(M) 3b1 1.4 × 10⁵ 1.8 × 10⁻³ 7.8 × 10⁷ 1.3 × 10⁻⁸ 3b3 1.6 × 10⁵ 1.2× 10⁻³ 1.3 × 10⁸ 7.5 × 10⁻⁹ 3b4 8.6 × 10⁴ 4.1 × 10⁻³ 2.1 × 10⁷ 4.8 ×10⁻⁸ 3b9 8.1 × 10⁴ 1.1 × 10⁻³ 7.4 × 10⁷ 1.4 × 10⁻⁸ MT4 3.4 × 10⁴ 1.5 ×10⁻³ 2.3 × 10⁷ 4.4 × 10⁻⁸

TABLE 1 (MN Strain) Fab K_(on) (^(M-1S-1)) K_(off)(S⁻¹) K_(a)(M⁻¹)K_(d)(M) 3b1 1.4 × 10⁵ 1.8 × 10⁻³ 7.8 × 10⁷ 1.3 × 10⁻⁸ 3b3 1.6 × 10⁵ 1.2× 10⁻³ 1.3 × 10⁸ 7.5 × 10⁻⁹ 3b4 8.6 × 10⁴ 4.1 × 10⁻³ 2.1 × 10⁷ 4.8 ×10⁻⁸ 3b9 8.1 × 10⁴ 1.1 × 10⁻³ 7.4 × 10⁷ 1.4 × 10⁻⁸ MT4 3.4 × 10⁴ 1.5 ×10⁻³ 2.3 × 10⁷ 4.4 × 10⁻⁸

In analyzing the data in Table 1 and Table 2, respectively, the bindingaffinity analysis of the randomized Fabs to gp120 from MN and IIIbstrains of HIV-1, the binding affinity of the randomized Fabs isenhanced as compared to the original gp120-specific Fab, MT4, from whichthe randomized Fabs were derived. Only one Fab, 3b4, did not exhibit anincrease in K_(a) in binding to gp120 from the MN strain as shown inTable 1. All other Fabs in binding to MN-derived gp120 had a three toten-fold increase in binding affinity over that of MT4. All therandomized Fabs showed even greater affinity to the IIIb-derived gp120as shown in Table 2. Fab 3b3 had a K_(a) of ten-fold greater than MT4 asit did with the MN-derived gp120. However, the binding affinity of 3b3to the MN versus the IIIb strain was also 10 fold higher, 1.3×10⁸ versus1.3×10⁹, respectively.

Thus, not only did the Fabs having randomized CDR1 and CDR3 exhibitheightened binding affinity as compared to the original MT4 Fab, thebinding affinities were further enhanced depending on the HIV-1 strainfrom which the gp120 ligand was derived. The randomization of the CDR1and CDR3 amino acid residue sequences in the four Fabs, 3b1, 3b3, 3b4and 3b9, therefore has resulted in a significant and unexpectedaugmentation of the binding affinity of the Fabs to gp120, from both MNand IIIb strains of HIV-1. This functional aspect is an importantattribute for diagnostic and therapeutic uses in that heightened bindingaffinity in comparison to known gp120-specific antibodies, as well asother HIV-1 antibodies, will provide an enhancement to targeting andneutralizing functions as described below.

2) Neutralizing Activity of gp120-Specific Fabs Having Randomized CDR1and CDR3

Binding of antibodies to viruses can result in loss of infectivity orneutralization and, although not the only defense mechanism againstviruses, it is widely accepted that antibodies have an important role toplay. However, understanding of the molecular principles underlyingantibody neutralization is limited and lags behind that of the othereffector functions of antibody. Such understanding is required for therational design of vaccines and for the most effective use of passiveantibody for prophylaxis or therapy. This is particularly urgent for thehuman immunodeficiency viruses.

A number of studies have led to the general conclusion that viruses areneutralized by more than one mechanism and the one employed will dependon factors such as the nature of the virus, the epitope recognized, theisotype of the antibody, the cell receptor used for viral entry and thevirus:antibody ratio. The principle mechanisms of neutralization can beconsidered as aggregation of virions, inhibition of attachment of virusto cell receptor and inhibition of events following attachment such asfusion of viral and cellular membranes and secondary uncoating of thevirion. One of the important features of the third mechanism is that itmay require far less than the approximately stoichiometric amounts ofantibody expected for the first two mechanisms since occupation of asmall number of critical sites on the virion may be sufficient forneutralization. For instance it has been shown that neutralization ofthe influenza A virion obeys single hit kinetics as described by Outlawet al., Epidemiol. Infect., 106:205-220 (1992).

Intensive studies have been carried out on antibody neutralization ofHIV-1. For review, see Nara et al., FASEB J., 5:2437-2455 (1991). Mosthave focussed on a single linear epitope in the third hypervariabledomain of the viral envelope glycoprotein gp120 known as the V3 loop.Antibodies to this loop are suggested to neutralize by inhibiting fusionof viral and cell membranes. Binding to the loop resulting inneutralization can occur prior to virus-cell interaction or followinggp120 binding to CD4. See, Nara, In Retroviruses of Human Aids andRelated Animal Diseases, eds. Girard et al., pp. 138-150 (1988); Linselyet al., J. Virol., 62:3695-3702 (1988); and Skinner et al., J. Virol.,67:4195-4200 (1988). Features of the V3 loop are sequence variabilitywithin the loop [Goudsmit et al., FASEB J., 5:2427-2436 (1991) andAlbert et al., AIDS, 4:107-112 (1990)] and sensitivity of neutralizingantibodies against the loop to sequence variations outside the loop[Nara et al., FASEB J., 5:2437-2455 (1991); Albert et al., AIDS,4:107-112 (1990); McKeating et al., AIDS, 3:777-784 (1989); and Wahlberget al., AIDS Res. Hum. Retroviruses, 7:983-990 (1991). Hence anti-V3loop antibodies are often strain specific and mutations in the loop invivo may provide a mechanism for viral escape from antibodyneutralization.

Recently considerable interest has focussed on antibodies capable ofblocking CD4 binding to gp120. A number of groups have described thefeatures of these antibodies as (a) reacting with conformational i.e.,non-linear epitopes, (b) reacting with a wide range of virus isolatesand (c) being the predominant neutralizing antibodies in humans afterlonger periods of infection. See, Berkower, et al., J. Virol.,65:5983-5990 (1991); Steimer et al., Science, 254:105-108 (1991); Ho etal., J. Virol., 65:489-493 (1991); Kang et al., Proc. Natl. Acad. Sci.,USA, 88:6171-6175 (1991); Posner et al., J. Immunol., 146:4325-4332(1991); and Tilley et al., Res. Virol., 142:247-259 (1991).

Neutralizing antibodies of this type would appear to present a promisingtarget for potential therapeutics. The mechanism(s) of neutralization ofthese antibodies is unknown although there is some indication that thismay not be blocking of virus attachment since a number of mousemonoclonal antibodies inhibiting CD4 binding to gp120 are eithernon-neutralizing or only weakly neutralizing.

The generation of human monoclonal antibodies against the envelope ofHIV-1 as described by Burton et al., Proc. Natl. Acad. Sci., USA,88:10134-10137 (1991) using combinatorial libraries allows a novelapproach to the problem of neutralization. Given the lack of athree-dimensional structure for gp120 and the complexity of the virus,the approach of enhancing the functional activity of gp120-specific Fabsthrough randomization of the CDR in either the heavy chain, light chainor both, seeks to explore neutralization at the molecular level throughthe behavior of related antibodies. Neutralization studies wereperformed as described herein on the human recombinant Fabs prepared inExample 1F and analyzed for binding affinity to gp120, derived fromeither MN or IIIb strains as described above.

a) Neutralizing Activity of gp120 specific Fabs Having Randomized CDR1and CDR3 with MN-derived gp120

A syncytium assay, was performed to measure neutralization ability ofthe recombinant human HIV-1 immunoreactive Fabs. For some of theseassays, the recombinant Fabs were first purified. One liter cultures ofSB containing 50 ug/ml carbenicillin and 20 mM MgCl₂ were inoculatedwith appropriate clones and induced 7 hours later with 2 mM IPTG andgrown overnight at 30 C. The cell pellets were sonicated and theresultant supernatant were concentrated to a 50 ml volume. The filteredsupernatants were loaded on a 25 ml protein G-anti-Fab column, washedwith 120 ml buffer at a rate of 3 ml/minute and eluted with citric acidat pH 2.3. The neutralized fractions were then concentrated andexchanged into 50 mM MES at pH 6.0 and loaded onto a 2 ml Mono-S columnat a rate of 1 ml/minute. A gradient of 0-500 mM NaCl was run at 1ml/minute with the Fab eluting in the range of 200-250 mM NaCl. Afterconcentrating, the Fabs were positive when titered on ELISA againstgp120 and gave a single band at 50 kD by 10-15% SDS-PAGE. Concentrationwas determined by absorbance measurement at 280 nm using an extinctioncoefficient (1 mg/ml) of 1.4.

A quantitative neutralization assay with the MN strain of HIV-1 wasperformed as described by Nara et al., AIDS Res. Human Retroviruses,3:283-302 (1987), the disclosure of which is hereby incorporated byreference. Monolayers of CEM-SS target cells were cultured with virus,in the presence or absence of Fab antibody, 3b1, 3b3, 3b4, 3b9 and MT4,and the number of syncytia forming units determined 3-5 days later. Anequivalent amount of virus was used in the assays to allow directcomparison of the various antibody concentrations tested. The assayswere repeatable over a virus-surviving fraction range of 1 to 0.001within a 2 to 4-fold difference in the concentration of antibody(P<0.001).

Assays were generally repeated at least twice with reproducible results.For the data reported in Table 3, the data is expressed as both IC₅₀(M⁻¹) and as Neutralization Titer in nanograms/milliliter (ng/ml). Theneutralization titer is calculated as 1/IC₅₀×(5×10¹⁰). The originalgp120-specific Fab, originally selected from a bone marrow library froman HIV-1 seropositive individual, was previously characterized by Barbaset al., J. Mol. Biol., 230:812-823 (1993) as having high bindingaffinity and equally effective neutralization ability in both syncytialformation and p24 assays. In the neutralization assays performed asdescribed herein, the MT4 Fab exhibited a neutralization titer ofapproximately 300 ng/ml to inhibit the infectivity of HIV-1 into thecells as measured by the decrease in syncytium formation. In strikingcontrast, four randomized Fabs of this invention, 3b1, 3b3, 3b4 and 3b9,all having been derived from original clone pMT4-3, exhibitedneutralization titers in this assay ranging from approximately 5 up to20 ng/ml. This represents a significant improvement of greater than 10fold increase in titer of the neutralizing ability of the randomizedFabs as compared to MT4, and antibody known to neutralize HIV-1infection.

TABLE 3 Neutralization Fab IC₅₀(M⁻¹) Titer (ng/ml) 3b1 5.42 × 10⁹ 9.23b3 9.02 × 10⁹ 5.5 3b4 2.57 × 10⁹ 19.4 3b9  6.4 × 10⁹ 7.8 MT4 1.69 × 10⁸296.0

Thus, the methods of this invention in randomizing both the CDR1 andCDR3 of the heavy chain of a clone which originally was effective atbinding to gp120 and neutralizing HIV-1 infection has resulted in thesignificant and unexpected improvement of Fabs that have heightenedbinding affinities as well as neutralization of infection capacities.Moreover, a correlation between the increase in binding affinity withthe ability to inhibit HIV-1 infection of cells exists as graphicallyshown in FIG. 3. In that figure, the binding affinity of the randomizedFabs along with MT4 was plotted against the neutralization titer asshown by IC₅₀. Five separate squares are shown in the figure for each ofthe five Fabs plotted based on the two functional characterizations. Alinear relationship is readily apparent in viewing the graph. Thus,there is a correlation of binding affinity to the ability to neutralizeHIV-1 infection. Moreover, all four randomized Fabs of this invention,exhibited enhanced correlations as compared to MT4 as shown on thefigure where the Fabs are shown in increasing linearity, with MT4 havingthe lowest binding affinity and neutralizing capacity, followed by 3b4,3b9 and 3b1 (comparably similar), and lastly 3b3. The latter Fab boundto gp120, derived from either MN or IIIb strains, and had the lowestneutralization titer of all randomized Fabs of this invention. Thus,randomization of the CDR1 and three CDR3 amino acid residues, as thetryptophan in the sixth position in the latter was conserved throughoutselection, resulted in the significant increase of both binding affinityand ability to neutralize HIV-1 infection compared to a non-randomizedgp120-specific Fab.

b) Neutralizing Activity of gp-120 Specific Fabs Having Randomized CDR1and CDR3 with IIIb-Derived gp120

A quantitative neutralization assay with the IIIb strain of HIV-1 wasperformed as described in Example H2a. For the results reported in Table4, the data is expressed as both IC₅₀ (M) and as Neutralization Titer innanograms/milliliter (ng/ml). The Neutralization Titer is calculated as1/IC₅₀×(5×10¹⁰). The gp120-specific Fab MT4, originally selected from abone marrow library from an HIV-1 seropositive individual, waspreviously characterized by Barbas et al., J. Mol. Biol., 230:812-823(1993), as having high binding affinity and equally effectiveneutralization ability in both syncytial formation and p24 assays. Inthe neutralization assays performed as described herein with gp120 fromthe IIIb strain of HIV-1, the MT4 Fab had a neutralization titer ofapproximately 39 ng/ml required to inhibit the infectivity of HIV-1 intothe cells as measured by the decrease in syncytium formation. Fourrandomized Fabs of this invention, 3b1, 3b3, 3b4 and 3b9, all havingbeen derived from original clone pMT4-3, had neutralization titersranging from approximately 22 to 66 ng/ml. This represents a clusteringof Fabs with similar potencies. With the gp120 from the IIIb strain ofHIV-1, a range of reactivity of only 3-fold was noted with the mostpotent Fab, 3b1, showing a modest 2-fold increase in potency whencompared to the original MT4 Fab.

TABLE 4 Neutralization Fab IC₅₀(M) Titer (ng/ml) 3b1 4.4 × 10⁻¹⁰ 22 3b39.4 × 10⁻¹⁰ 47 3b4 9.9 × 10⁻¹⁰ 50 3b9 1.3 × 10⁻⁹  66 MT4 7.7 × 10⁻¹⁰ 39

While the Fabs of this invention, 3b1, 3b3, 3b4 and is 3b9, do notdemonstrate the same striking increase in potency with IIIb as wasdemonstrated with MN, it should be noted that the MT4 Fab from whichthese Fabs were derived demonstrated a 10-fold greater potency withIIIb-derived gp120 than with MN-derived gp120.

The kinetics of binding of purified Fab to two types of gp120 werecompared from the highly divergent isolates MN and IIIb. Myers et al.,Human Retroviruses and Aids 1992, Theoretical Biology and Biophysics,Los Alamos, N.M., (1992). A comparison of the recombinant proteinMN-derived gp120 and IIIb-derived gp120 revealed 88 amino acid residuechanges in the aligned sequences as well as 11 deletions and 5insertions of amino acid residues. Infectivity of a target cell requiresbinding of the viral surface glycoprotein gp120 to the CD4 molecule onthe surface of the target, therefore, the CD4 binding site on gp120 is acommon target for anti-viral antibodies. Sun et al., J. Virol.,63:3579-3585 (1989); Thali et al., J. Virol., 65:6188-6193 (1991);Tilley et al., Res. Virol., 142:247-259 (1991); Karwowska et al., AIDSRes. Hum. Retroviruses, 8:1099-1106 (1992); and Moore et al., J. Virol.,67:863-875 (1993). However, antibodies to this region are not generallyparticularly potent in terms of virus neutralization. Furthermore, suchantibodies tend to be even less potent against primary isolates of virusthan the more commonly employed laboratory strains. Moore et al.,Perspectives in Drug Discovery and Design, 1:235-250 (1993). Therefore,an HIV-1 neutralizing human antibody directed against the CD4-bindingsite of gp120 which demonstrates increased affinity, potency, andbroadened strain reactivity would be highly desirable in prophylacticand therapeutic applications.

The Fabs of this invention demonstrate exceptional potency in the 10⁻⁹range with both the gp120 derived from the MN and IIIb laboratorystrains of HIV-1. The Fab 3b3 was selected for further study inneutralization assays with primary clinical (“field”) isolates due toits 54-fold improvement in affinity to MN-derived gp120.

c) Neutralizing Activity of gp120-Specific Fab 3b3 Having a RandomizedCDR1 and CDR3 with Primary Clinical HIV Isolates

The key issue in producing antibodies to HIV-1 for therapeutic orprophylactic purposes is that they should be highly potent (of highaffinity and neutralizing ability) and be cross reactive with a widerange of primary clinical (field) isolates. These are generally twoopposing characteristics. The degree of antigenic relatedness betweendifferent patient isolates of HIV has been examined bycross-neutralization in a microplaque assay. Wrin et al., J. of AcquiredImmune Deficiency Syndromes, 7:211-219, (1994). The cross-neutralizationassays with heterologous sera and virus isolates from 11 individualsrevealed variations in breadth of neutralization among individual seraand variation in the frequency of neutralization among the differentprimary clinical isolates.

A quantitative assay to measure the reduction of infectivity of primaryclinical isolates of HIV-1 in the presence of the Fabs MT4 and 3b3 wasdetermined in a microplaque assay as described in Hanson et al., J. ofClin. Microb., 2030-2034 (1990). Primary clinical isolates of HIV-1 wereisolated from frozen peripheral blood lymphocytes obtained fromseropositive donors as described in Gallo et al., J. of Clin. Microb.,1291-1294 (1987) and cultivated in peripheral blood mononuclear cells(PBMC). Briefly, HIV isolates were obtained by incubating frozenHIV-infected patient PBMCs with seronegative donor PBMCs in RPMI-1640medium containing 20% heat-inactivated fetal bovine serum, 2 ug/mlpolybrene, 5% interleukin-2, and 0.1% anti-human leukocyte interferon.The cultures were fed with fresh donor PBMCs once a week, and thesupernatants were assayed for the presence of reverse transcriptase (RT)activity beginning at day 11. The cultures were considered positive if,for 2 consecutive weeks, the RT counts were >10-fold higher than thosein the cultures of the seronegative donor PBMCs alone.

The resultant RT-positive virus isolates were tested for cytolysis inthe MT2 (α-4 clone) (Hanson et al., J. of Clin. Microb., 2030-2034,1990), and the viruses which were found to cytolytic, a requirement forviruses usable in the subsequent MT2 microplaque assay system.Supernatant fluids from the primary PBMC isolation cultures were used toinfect expanded cultures of phytohemagglutinin (PHA)-stimulated PBMCsfrom healthy seronegative blood donors. These infected PBMC cultureswere grown in RPMI-1640 medium supplemented with 15% fetal bovine serum,5% interleukin-2, 0.1% anti-α interferon, 2 ug/ml polybrene, 50 ug/mlgentamicin, 100 U/ml penicillin, and 100 ug/ml streptomycin. The crudesupernatants were harvested after 7 days and frozen as viral stocks at−70° C.

The primary clinical isolates of HIV-1 used in this microplaque assayare designated VL135, VL263, VL596, VL069, VL434, VL114, VL172, VL530,and VL750. Isolates VL135, VL434, VL069, VL263, and VL596 have beenpreviously described as isolates 1, 3, 4, 5, and 7, respectively, inWrin et al., J. of Acquired Immune Deficiency Syndromes, 7:211-219(1994).

The laboratory HIV-1 strains MN and IIIb as well as isolate VL069 werepropagated in H9 cells as controls in the microplaque assay. Propagationof VL069 in H9 cells was performed to illustrate a host cell effectwhich results in a sensitization when isolates are propagated in H9cells and has been previously described in Sawyer, et al., J. Virol.,68:1342-1349 (1994).

The Fabs MT4 and 3b3 and a pool of human plasma from 13 HIV-1seropositive patients (+PHP) were used as the source of neutralizingantibodies in a 96-well microtiter plaque reduction assay as describedby Hanson et al., J. of Clin. Microb., 2030-2034 (1990). Briefly, serialdilutions of the Fabs MT-4 or 3b3 (starting at 50 ug/ml and decreasing)or heat-inactivated pooled patients' plasma (starting at a 1:10 dilutionand decreasing to 1:256) were combined with an equal volume containing10-25 plaque-forming units (PFU) of HIV per well and incubated for 18hours at 37 C. The diluent used for both virus and patient plasmadilutions contained 50% normal human serum pool (prepared byrecalcification of human plasma) which had been heat inactivated at 56C. for 60 minutes to remove complement. Negative control wells alsocontained 50% normal human serum pool with no patient immune serum.After the 18 hour incubation of Fabs or serum and virus, 90,00.0 MT2cells were added per well and incubated at 37 C. for 1 hour. SeaPlaqueAgarose in assay medium at 39.5 C. was then added to a finalconcentration of 1.6%. While the warm agarose was still molten, themicrotiter plates were centrifuged at 20 C. for 20 minutes at 500×g toform cell monolayers. The plates were incubated for 5 days at 37 C. andthen stained 18 to 24 hours with 50 ug/ml propidium iodide. Thefluorescent plaques were counted with transillumination by a 304 nmultraviolet light source using a low-power stereo zoom microscope.Inhibition of infectivity, or neutralization titer, is defined as theug/ml of Fab or the plasma dilution giving 50% inhibition of plaquecount as compared with controls. Within an experimental run, theintrinsic statistical error of the interpolated titers averages±30%.

The inhibition of infectivity, or neutralization titer, for the Fabs MT4and 3b3 and the pooled HIV seropositive human plasma from 13 donors(+PHP) is given in Table 5. The neutralization titer for each of theviral isolates is expressed as the minimum ug/ml of Fabs MT4 and 3b3required for 50% inhibition of plaque count as compared to the controls.The neutralization titer for each of the viral isolates is expressed asthe minimum titer of the pooled HIV seropositive human plasma from 13donors (+PHP) required for 50% inhibition of plaque count as compared tothe controls.

The Fab MT4 was able to neutralize only two of the nine primary clinicalisolates assayed at concentrations of 50 ug/ml and less as measured asthe ug/ml required for 50% inhibition of plaque count as compared to thecontrols. In contrast, the Fab 3b3 was able to neutralize six of thenine primary clinical isolates at concentrations of 50 ug/ml and less asmeasured as the ug/ml required for 50% inhibition of plaque count ascompared to the controls. Thus, the highest affinity Fab, 3b3, was ableto neutralize an additional four primary clinical isolates as comparedto the Fab MT4 from which it was derived. Fifty-percent neutralizationof isolates VL135 and VL530 by Fab 3b3 at 38.9 and 29.5 ug/ml,respectively, is significant because the Fab MT4, from which 3b3 wasderived, showed insignificant levels of neutralization (about 10%) at 50ug/ml. Neutralization of MN- and IIIb-derived gp120 was improvedapproximately 5-fold in the microplaque assay when compared to thepreviously performed syncytium formation assay described in Example 2H.

TABLE 5 Neutralization of Field Isolates of HIV ug/ml required for 50%neutralization Titer Virus Host Cell MT-4 3b3 +PHP VL155 PBMC >50 38.91:33 VL263 PBMC 17.0 6.6 <1:10 VL596 PBMC 33.1 17.0 1:10 VL069PBMC >50 >50 <1:10 VL434 PBMC >50 10.5 1:10 VL114 PBMC >50 5.2 <1:10VL172 PBMC >50 >50 1:10 VL530 PBMC >50 29.5 <1:10 VL750 PBMC >50 >501:10 IIIb H9 0.36 0.068 1:767 MN H9 0.18 0.044 1:24,000 VL069 H9 3.6 3.51:1,200

Thus, the methods of this invention in randomizing both the CDR1 andCDR3 of the heavy chain of an Fab clone which originally was effectiveat binding to gp120 and neutralizing HIV-1 infection has also resultedin the significant and unexpected improvement of Fabs that havebroadened neutralization activities. This broadened neutralizationactivity was first demonstrated by an increase in binding affinitieswith the highly divergent HIV-1 gp120 isolates MN and IIIb. Selectivepressure during the panning process could have been applied to favorcross-reactivity by selecting with a mixture of divergent gp120s,however, this did not prove to be necessary as Fabs with increasedbinding affinity to both MN and IIIb-derived gp120s were identified.

Potencies as judged by quantitative infectivity in neutralization assayswith MN- and IIIb-derived gp120 stocks are improved as well. Affinity iswell correlated with neutralizing ability with the MN-derived gp120. Thepotencies of the Fabs of this invention are equivalent to the potenciesof soluble CD4 (sCD4). Layne et al., Nature, 346:277-279, (1990). Thisability to neutralize with the potencies equivalent to sCD4 is unique.

The broadened neutralization activities of the Fabs of this inventionwere further demonstrated in quantitative neutralization assays withprimary clinical isolates of HIV. The Fab 3b3, which demonstrated thehighest affinity to MN-derived gp120, is able to neutralize anadditional four primary clinical isolates as compared to the Fab MT4from which it was derived. Characterization of these primary clinicalisolates in neutralization assays revealed patterns of heterologousneutralization that suggested multiple phenotypes (Wrin et al., J. ofAcquired Immune Deficiency Syndromes, 7:211-219, 1994). Thus, therandomization of CDR1 and CDR3 of the heavy chain of an Fab whicheffectively binds gp120 and neutralizes HIV-1 infection resulted in Fabswith broadened neutralization reactivity with primary clinical isolatesof multiple phenotypes.

2. Preparation of Four Phagemid Libraries Having Randomized Heavy andLight Chain CDR and Selection of Affinity-Optimized Fabs ExpressedTherefrom

A. Overview of the Methods to Obtain Randomized Heavy and Light Chaingp120-Specific Fab Antibodies

Mutagenesis of heavy chain CDR1 and CDR3 regions of pMT4-3 as describedin Example 1 resulted in producing a phagemid, designated p3b3, forexpressing the 3b3 heavy and light chain Fab heterodimer antibody whichdemonstrated an increase in affinity for gp120 with broadenedneutralization reactivity with primary clinical isolates of multiplephenotypes.

The methods of producing higher affinity gp120-specific Fab antibodiesof this invention, as described in Examples 2 and 3, are based on theCDR-directed random mutagenesis of either pMT4-3 or p3b3 obtained inExample 1. More particularly, preselected CDR were randomized intemplate DNA to optimize binding to the substrate gp120. Following theselective optimization, nucleotide fragments encoding mutagenized CDRfrom different gp120-reactive Fabs were then combined in particularcombinations to form composite heavy and light chain domains that weresubsequently inserted into an expression vector for the expression ofsoluble composite optimized CDR-containing Fabs having dissociationconstants (K_(d)) of 10⁻¹⁰M or greater.

The following general approach was used to obtain composite optimizedCDR-containing Fabs having enhanced affinities: 1) New phagemidlibraries were generated using the methods of this invention where eachlibrary resulted from amplification coupled with random mutagenesis of aparticular CDR in preselected phagemid templates; 2) Following screeningof the phagemid-Fab displayed libraries on gp120, particular clones wereselected for preparation of additional libraries to obtain clones havingmultiple optimized CDR for conferring high affinity Fab antibodyinteraction with gp120; 3) Clones expressing preferred mutagenized Fabswere then used to create unique Fab-expressing clones having randomizedand selected CDR1 and CDR3 heavy and light chains optimized for highaffinity binding to gp120.

In producing and screening libraries as described in the overview above,the following libaries were prepared from which gp120-specific cloneswere selected for sequence and affinity analysis of expressed Fabs.

For one library, the pMT4-3 library having randomized heavy chain CDR1(15 nucleotides) was subjected to a second round of screening asdescribed in Example 1 in order to obtain additional gp120-specificclones expressing Fabs that are not glycosylated by having a histidineresidue in the first position of CDR1 instead of aglycosylation-reactive asparagine residue as was obtained in the firstscreen in the clones encoding Fabs 3b1, 3b3, 3b4, and 3b9. The DNAencoding the selected Fabs was then inserted into the pPho-TT vector forthe preparation of soluble Fabs for affinity and nucleotide sequencedetermination.

For a second library, the heavy chain CDR3 of phagemid 3b3 in pComb3Hwas also separately randomized over 15 nucleotides contiguous to the 12nucleotides that were previously mutagenized in pMT4-3 for forming aphagemid expressing Fab 3b3 as described in Example 1. This extension ofthe mutagenized area in CDR3 was performed to obtain Fabs havingaffinities for gp120 equal to or greater than that obtained with Fab3b3. Amplification products from the overlap PCR containing the newlyrandomized CDR3 were ligated back into 3b3 to form a randomized libraryhaving a randomized heavy chain CDR3 along with the previouslyrandomized and selected heavy chain CDR1 and the nonmutagenized lightchain originally derived from pMT4-3. Expression, selection andcharacterization were performed as described above.

For a third library, the light chain CDR1 of phagemid 3b3 in pComb3H wasseparately randomized over 18 nucleotides that encode six of the 12amino acids in CDR1. The resultant amplification products havingrandomized light chain CDR1 were then ligated into 3b3 to form a3b3-based light chain CDR1 mutagenized library. Thus, selected clonesfrom this library contained the nucleotide sequences for expressing theoriginal 3b3-mutagenized heavy chain CDR1 and CDR3 and the newlymutagenized light chain CDR1.

A fourth library in which a portion (15 nucleotides) of the light chainCDR3 were randomized was prepared as described for the third libraryabove. The resultant 3b3-based library contained clones havingnucleotide sequences for expressing the 3b3 mutagenized heavy chain CDR1and CDR3 and the newly mutagenized light chain CDR3 with the original3b3 light chain CDR1. Expression, selection and characterization wereperformed as described above.

To create gp120-binding synthetic Fabs having optimized light chain CDR1and CDR3, a preferred clone, phagemid D, was selected from the fourthlibrary having a randomized and selected light chain CDR1 and then wassubjected to another round of mutagenesis on the light chain CDR3. Forthe resultant library, the expression and panning process was performedby two different methods, specific elution with acid versus competitionwith soluble Fab 3b3 followed by elution with acid. The latter elutionprocedure resulted in the isolation of clones encoding Fabs with bothlight- and heavy-chain mutagenized CDR1/CDR3 that exhibited a K_(d) of10⁻¹¹M or greater.

Individual light and heavy chain sequences having composite CDRsprovided by separate identified and characterized Fab-expressing cloneswere prepared to eventually create unique Fabs having affinity optimizedheavy and light chain variable domains compared to the clones from whichthe CDR were obtained.

One of skill in the art will appreciate that the above-enumerated stepscan be performed in the order given or in a different order to prepareFabs with mutagenized heavy and light chain CDR regions. The preparationof the aforementioned libraries and selection of gp120-specific Fabstherefrom are presented in detail below.

1) Preparation of pComb3H Expression Vector

The expression vector, pComb3H, is a modified version of the originalpComb3 phagemid expression vector that was described in Example 1. Aswith pComb3, pComb3H also provides for the expression of phage-displayedbacteriophage coat protein 3-anchored proteins. Gene III of filamentousphage encodes the 406-residue minor phage coat protein, cpIII (cp3),which is expressed prior to extrusion in the phage assembly process on abacterial membrane and accumulates on the inner membrane facing into theperiplasm of E. coli. The nucleotide and amino acid residue sequences ofgene 3 and the encoded coat protein, respectively, are familiar to oneof ordinary skill in the art and have been published in InternationalApplication WO 92/18619, the disclosure of which is hereby incorporatedby reference.

The modified vector, pComb3H, was designed to provide a human consensussequence to the amino terminus of the heavy chain as described below.Furthermore, all homologous regions found in the original pComb3 wereremoved to increase the stability of the vector. The pComb3H vector fromthe lacZ promoter through the Not I restriction site is illustrated inFIG. 6 and the nucleotide sequence of the complete vector including thenucleotide sequences encoding the anti-tetanus toxin Fab, occupying theheavy and light chain cassettes, is listed in SEQ ID NO 43. The pComb3Hvector containing the anti-tetanus toxin (TT) encoding sequences isdesignated pComb3H-TT. When the TT-encoding stuffers are removed fromthe pComb3H-TT vector, preselected heavy and light chain nucleotidesequences of this invention can be directionally inserted.

In using pComb3H, the first cistron encoding a periplasmic secretionsignal (ompA leader) is operatively linked to a kappa light chain. Thenucleotide sequence of the ompA, a leader sequence for directing theexpressed protein to the periplasmic space, was as reported by Skerra etal., Science, 240:1038-1041 (1988). The second cistron encoded a pelBleader operatively linked to the fusion protein, Fd-cpIII. The presenceof the pelB and ompA leaders facilitated the coordinated but separatesecretion of both the fusion protein comprising the Fd/cp3 fusionprotein and the kappa light chain from the bacterial cytoplasm into theperiplasmic space.

Each chain was delivered to the periplasmic space by the pelB or ompAleader sequence, which was subsequently cleaved. The heavy chain wasanchored in the membrane by the cp3 membrane anchor domain while thelight chain was secreted into the periplasmic space. Fab molecules wereformed from the binding of the anchored heavy chains with the solublelight chains.

The pComb3H vector was derived from the pComb3 vector and contains alacZ promoter, ribosome binding site, ColE1 and f1 origins, and abeta-lactamase gene which were derived from pBluescript which haspreviously been described by Short et al., Nuc. Acids Res. 16:7583-7600(1988). The complete nucleotide sequence of pBluescript has the GenBankAccession Number 52330. The DNA encoding amino acid residues 230 to 406of cp3 was inserted following the Sfi I restriction site which is 3′ ofthe Spe I restriction site (FIG. 6).

The pComb3H vector contains two Sfi I restriction sites (FIG. 6). One ofthe Sfi I sites is contained within the nucleotide sequence encoding theompA leader which directs the secretion of the light chain and istherefore located 5′ of the Sac I site. The second Sfi I site is betweenthe Spe I site and the cp3 membrane anchor sequence. When the pComb3Hvector contains DNA encoding a light chain and Fd, the two Sfi I sitesare respectively located at the 5′ end of the DNA sequence encoding thelight chain and the 3′ end of the DNA sequence encoding the Fd.Digestion of the pComb3H vector containing DNA encoding heavy and lightchains with Sfi I removes a cassette comprising the DNA encoding thelight chain, translational stop sequences, ribosomal binding site, pelBleader, and Fd. This cassette can then be directionally inserted intoanother expression vector, such as the pPho-TT vector, described in thisinvention. Therefore, in addition to the original restriction sites inpComb3, the addition of the Sfi I restriction sites is anothermodification to allow for subcloning of the entire heavy and light chaincassette in one fragment into an expression vector designated pPho forexpression of soluble Fabs as described below.

Thus, the resultant combinatorial vector, pComb3H, consisted of a 3394base pair DNA molecule having two cassettes to express one fusionprotein, Fd/cp3, and one soluble protein, the light chain. The vectoralso contained nucleotide sequences for the following operatively linkedelements listed in a 5′ to 3′ direction: a first cassette consisting ofLacZ promoter/operator sequences; an Eco RI restriction site; a ribosomebinding site; an ompA leader; a Sfi I restriction site; a spacer region;a cloning region bordered by 5′ Sac I and 3′ Xba I restriction sites; aNco I restriction site located between the two cassettes; and a secondcassette consisting of an expression control ribosome binding site; apelB leader; a human consensus amino terminus spacer region encoding theamino acid residues Glu-Val-Gln-Leu-Leu-Glu (SEQ ID NO 44); a cloningregion bordered by 5′ Xho I and a 3′ Spe I restriction sites followed bya Sfi I restriction site; the sequences encoding bacteriophage cp3followed by a stop codon and a Nhe I restriction site; expressioncontrol stop sequences and a Not I restriction site.

The pComb3H vector sequence as given in SEQ ID NO 43, and designatedpComb3H-TT, contains a light chain TT-encoding stuffer that is 1,200 bpin length and a heavy chain TT-encoding stuffer that is 300 bp inlength. For cloning light chain variable domains for expressing Fabs ofthis invention, the light chain stuffer of an anti-tetanus toxin Fablight chain was removed by digestion with the restriction enzymes Sac Iand Xba I prior to insertion of a similarly digested light chain. Forcloning heavy chain variable domains for expressing Fabs of thisinvention, the heavy chain stuffer of an anti-tetanus toxin Fab heavychain was removed by digestion with the restriction enzymes Xho I andSpe I prior to insertion of a similarly digested heavy chain.

The cassette containing DNA encoding the light chain sequences requiredfor the expression of the heavy chain and DNA encoding the heavy chainwere then removed from the pComb3H vector as described below anddirectionally inserted into the pPho-TT vector for the production ofsoluble Fab by digestion with the restriction enzyme Sfi I.Alternatively, the pComb3H vector can be digested with the restrictionenzymes Nhe I and Spe I and religated to remove the cp3 membrane anchorsequence and express a soluble Fab.

2) Preparation of pComb3H containing the Phagemid 3b3 Heavy and LightChain Variable Domains

In order to obtain CDR randomized expressed synthetic human Fabantibodies having both heavy and light chain variable domains, the heavyand light chain variable domain sequences of phagemid 3b3 weredirectionally ligated into pComb3H-TT by sequential replacement of eachTT stuffer. This pComb3H-3b3 containing phagemid vector, designatedpComb3H-3b3, was then used as a template for subsequent mutagenesisprocedures. The randomization of 3b3-derived Fabs are described inExamples 2C through 2E.

For preparation of pComb3H-3b3, the phagemid 3b3 was first digested withXho I and Spe I and the fragment was then ligated with a similarlydigested pComb3H-TT vector to form a pComb3H vector containing the heavychain variable domain of phagemid 3b3. To create a pComb3H phagemid forexpressing both heavy and light chains derived from 3b3, the light chaincassette of 3b3, was digested with Sac I and Xba I and then insertedinto the pComb3H-TT vector containing the 3b3 heavy chain that wassimilarly digested. In other words, a pComb3H-based vector containingthe nucleotide sequence encoding the 3b3 mutagenized heavy chainvariable domain and the nucleotide sequence encoding the 3b3-derivedlight chain variable domain respectively replaced the heavy and lightchain anti-tetanus toxin stuffers in pComb3H-TT. The 3b3 light chainvariable domain nucleotide sequence is the same as that of pMT4-3 asshown in FIG. 10 and in SEQ ID NO 62.

Following expression, the library of pComb3H-phage -anchored Fabs wasscreened by panning as previously described.

B. Preparation of Randomized CDR1 of the Heavy Chain Variable Domain ofPhagemid pMT4-3

The CDR1 of the heavy chain variable domain in pMT4-3 was mutagenized asdescribed in Example 1 and by Barbas et al., Proc. Natl. Acad. Sci.,USA, 91:3809-3813 (1994), the disclosure of which is hereby incorporatedby reference. However, all of the screened and selected gp120-specificFabs contained an asparagine (N) residue in the first position of CDR1.This is not a preferred residue as it serves as a glycosylation site.Therefore, to obtain additional Fabs having a preferred histidine (H)residue in that location, a second round of screening was performed onthe heavy chain CDR1 in pMT4-3 as described in Example 1. The nucleotidesequences encoding resultant gp120-reactive phage-displayed Fabs werethen subcloned into a pPho-TT vector as described below.

1) Amino Acid Residue Sequence Analysis of pMT4-3 Derived Fabs Having aRandomized Heavy Chain CDR1

Nucleic acid sequencing of the CDR1 randomized clones produced above wasperformed on six randomly chosen double-stranded Fab-expressing DNAclones using Sequenase 1.0 (USB, Cleveland, Ohio). Nucleic acidsequencing can be performed using any of the Fab-expressing vectorsdescribed in this invention as a template and is not dependent onwhether the vector-driven Fab expression is soluble or membraneanchored. The alignment of derived amino acid residue sequences with oneanother and with the Genbank database made use of the MacVector suite ofprograms. The derived heavy chain amino acid residue sequences of sixselected specific synthetic gp120-specific Fabs and MT4 are shown inFIGS. 7A and 7B. The alignment of the framework (FR) and complementaritydetermining regions (CDR) in the variable heavy chain domain as shown inFIG. 7 reveals that the original MT4 gp120-specific Fab obtained fromscreening a bone marrow library from an asymptomatic HIV-1 seropositiveindividual the amino acid residue sequence of CDR1 wasAsn-Phe-Val-Ile-His (SEQ ID NO 1, from residues 28-32). Sequencecomparisons indicated a preference for either asparagine (N) orhistidine (H) at position 31, an aromatic residue at position 32,primarily threonine (T) at position 33, either isoleucine (I) or leucine(L) at position 34, and hydrophobic and/or aromatic residues at position35.

The six amino acid residues indicated in FIGS. 7A and 7B for CDR1 havethe randomized and selected amino acid residue sequences from positions28-32 in each of the SEQ ID NOs indicated: H4H1-1: His-Phe-Thr-Val-His(SEQ ID NO 45); H4H1-3: His-Phe-Thr-Leu-His (SEQ ID NO 46); H4H1-5:His-Phe-Thr-Ile-Met (SEQ ID NO 47); H4H1-6: Asn-Tyr-Thr-Leu-Gln (SEQ IDNO 48); H4H1-7: Asn-Phe-Thr-Leu-Ile (SEQ ID NO 49); and H4H1-8:Asn-Trp-Thr-Ile-Met (SEQ ID NO 50). As shown in FIGS. 7A and 7B, threeof the six screened and selected heavy chain CDR1-mutagenized Fabscontained the preferred histidine residue as the first residue in CDR1to avoid glycosylation as occurs if asparagine was present as was thecase in MT4.

The DNA encoding the CDR1-mutagenized heavy chain Fabs selected forbinding to gp120 were then separately transferred to the vector pPho-TTfor expression of soluble Fabs. The soluble Fabs were then analyzed forbinding affinity to gp120.

2) Binding Affinity Analysis of gp120-Specific Fabs Having RandomizedHeavy Chain CDR1

The DNA that encoded Fabs containing a randomized heavy chain CDR1 whichhad been selected for binding to gp120 was then transferred to thevector pPho-TT for expression of soluble Fab. Affinity analysis of thebinding of soluble Fabs was then performed as described below. Thetransfer of the nucleotide sequences encoding the gp120-specific Fabscoding regions for this purpose was facilitated by the presence of Sfi Irestriction sites flanking the Fab coding regions in both the pComb3H-TTand pPho-TT vectors.

a) Preparation of pPho-TT Containing gp120-Specific Fabs

The expression vector, pPho-TT, is a modified version of the pComb3 andpComb3H phagemid expression vectors that were respectively described inExamples 1 and 2A. The pComb3 and pComb3H-TT vectors provided for theexpression of soluble Fabs by the removal of the phagemid gene 3 anchorsequence encoding cp3 from the expression vector as described in Example1G. As with the pComb3 and pComb3H vectors, pPho-TT also provides forthe expression of soluble Fabs which are secreted to the periplasmicspace. However, while expression of soluble Fabs from the pComb3 andpComb3H-TT vectors is regulated by the lacZ promoter, the expression ofsoluble Fabs from the pPho-TT vector is regulated by the alkalinephosphatase (phoA) promoter. As is well known to those of ordinary skillin the art, the phoA promoter is inducible under phosphate starvationconditions (Sambrook et al., in “Molecular Cloning: a LaboratoryManual”, 2nd edition, Cold Spring Harbor Laboratory Press (1989)).

The pPho-TT vector is identical to the pComb3H-TT vector from the Eco RIsite to the second Sfi I site, following the Spe I site. This region ofpComb3H-TT is illustrated in FIG. 6. The nucleotide sequence of pPho-TT,the complete pPho vector including nucleotide sequences encoding ananti-tetanus toxin Fab which occupy the heavy and light chain cassettes,is listed in SEQ ID NO 51.

In using pPho-TT, the first cistron is identical to that encoded by thepComb3H-TT first cistron and provides for the expression and secretionof a kappa light chain. The second cistron encoded a pelB leaderoperatively linked to the heavy chain protein, Fd. The presence of theompA and pelB leaders facilitated the coordinated but separate secretionof both the kappa light chain and Fd, respectively, from the bacterialcytoplasm into the periplasmic space.

The Fd and kappa light chains were secreted to the periplasmic space bytheir respective leader sequences which were then cleaved. Fab moleculeswere formed form the binding of the secreted Fd and kappa light chains.

The pPho-TT vector, a schematic restriction map of which is shown inFIG. 8, also contained a ribosome binding site, ColE1 and f1 origins,and a beta-lactamase gene as described for the pComb3H vector in Example2B1a). The pPho-TT vector contained Xho I and Spe I sites for thedirectional insertion of DNA encoding a heavy chain and Sac I and Xba Isites for the directional insertion of DNA encoding a light chain. Inaddition, the pPho vector contained two Sfi I sites, identical to thoseof the pComb3H vector to facilitate the transfer of the entire heavy andlight chain cassette in one fragment from the pComb3H vector into thepPho vector for the expression of soluble Fabs as herein described.Thus, the sites in the pPho vector allowed for the directional insertionof DNA encoding the heavy or light chain in two separate steps or forthe directional insertion of a single cassette comprising DNA encodingboth the heavy and light chain in a single step. When the DNA encodingthe heavy or light chain is inserted into the pPho-TT vector in separatesteps, the source of the inserted DNA can be from the same or differentFabs. In addition, a portion of either the heavy or light chainDNA-encoding sequence can be inserted into the pPho vector as describedfurther herein.

Thus, the pPho-TT vector consisted of a DNA molecule having twocassettes to express two soluble proteins, a heavy chain (Fd) and alight chain. The vector contained nucleotide residue sequences for thefollowing operatively linked elements listed in a 5′ to 3′ direction: afirst cassette consisting of the phoA promoter/operator sequences; anEco RI restriction site; a ribosome binding site; an ompA leader; a SfiI restriction site; a spacer region; a cloning region bordered by 5′ SacI and 3′ Xba I restriction sites; a Nco I restriction site locatedbetween the two cassettes and a second cassette consisting of anexpression control ribosome binding site; a pelB leader; a humanconsensus amino terminus spacer region encoding the amino acid residuesGlu-Val-Gln-Leu-Leu-Glu (SEQ ID NO 44); a cloning region bordered by 5′Xho I and 3′ Spe I restriction sites followed by a Sfi I site;expression control stop sequences, and a Not I restriction site.

The pPho-TT vector sequence, as given in SEQ ID NO 51, contains a lightchain stuffer that is 1,200 bp in length and a heavy chain stuffer thatis 300 bp in length. The nucleotide sequences of the heavy and lightchain stuffers encoded the heavy and light chain variable domains of atetanus toxin (TT)-specific Fab, respectively. Thus the pPho vectorsequence containing the light and heavy chain stuffers as given in SEQID NO 51 is designated, pPho-TT. For the insertion of heavy or lightchain variable domains for the expression of Fabs of this invention, theheavy and light chain stuffers of pPho-TT were removed as described inExample 2B1a) for the preparation of the pComb3H-TT vector forexpression of Fabs of this invention.

To prepare soluble heterodimers from pComb3H vectors as described inExamples 2C-2E, phagemid DNA encoding gp120-reactive clones was firstisolated and then digested with Sfi I. pPho-TT was similarly digested.Digestion of the phagemid DNA and pPho-TT with the Sfi I restrictionenzyme produced compatible cohesive ends. The 1.6-kb DNA fragmentcomprising the gp-120-reactive heavy and light chains and the 4.7-kb DNAfragment comprising the pPho vector were gel-purified (0.6% agarose) andligated together. Transformation of E. coli DH10B (BioRad, Richmond,Calif.) afforded the isolation of recombinants comprising DNA encodingthe gp120-reactive heavy and light chains and the pPho expressionvector. Clones were examined for insertion of the heavy and lightchain-encoding DNA fragment by Xho I - Xba I digestion, which shouldyield an 1.6-kb fragment. Thus, the DNA-encoding gp120-reactive heavyand light chains was transferred from the pComb3H to the pPho vector forthe expression of soluble Fabs.

To prepare soluble heterodimers from pComb3-based vectors as describedin Example 2B for vectors having a CDR1-randomized heavy chain, theheavy and light chain cassettes were separately isolated by digestionwith Xho I/Spe I and Sac I/Xba I, respectively, and then sequentiallydirectionally ligated into a similarly digested pPho-TT therebysequentially replacing the TT heavy and light chain stuffer fragments.

b) Expression and Purification of Soluble gp120-Specific Fabs from pPhoExpression Vectors

For the expression and purification of gp120-specific soluble Fabs,clones were grown in low-phosphate medium to induce the phoA promoter asis well known in the art and described in Sambrook et al., in “MolecularCloning: a Laboratory Manual”, 2nd edition, Cold Spring HarborLaboratory Press (1989)) and soluble Fabs purified as described inExample 1H2a).

c) Binding Affinity Analysis Using Plasmon Resonance

The second screened group of heavy chain CDR1-mutagenized Fabs selectedby panning against gp120 as described in Example 2B2) were expressed insoluble form from pPho as described above for use in affinitydeterminations with laboratory isolates of gp120. Surface plasmonresonance was performed as described in Example 1H to determine theaffinity of the selected Fabs having the preferred histidine residue inthe first position of heavy chain CDR1.

The results of the Fab binding affinity analysis as measured by theequilibrium dissociation constant (K_(d), M) of those Fabs having thepreferred histidine residue (H4H1-1, H4H1-3 and H4H1-5) to gp120 strainIIIb are listed in Table 6. All measurements in the subsequent tablesare presented as K_(d), M. In addition, fold increases of affinity arecalculated to provide a comparison of the effect on affinity ofmutagenizing preselected heavy or light chain CDR with that of thenonmutagenized Fab MT4 obtained from a patient. The fold increases arecalculated by dividing the measured K_(d) (6.3×10⁻⁹M) of the Fab MT4with each of the measured K_(d) of each new Fab having mutagenized CDR.

TABLE 6 ²Fold Increase of Mutant Fab ¹K_(d) K_(d) Compared to MT4 K_(d)H4H1-1 1.7 × 10⁻⁹ 3.7  H4H1-3 2.0 × 10⁻⁹ 3.15 H4H1-5 2.45 × 10⁻⁹  2.57H4H1-6 ³ND  ND H4H1-7 ND ND H4H1-8 ND ND ¹Dissociation constants ofmutants' binding to gp 120 (IIIB strain). ²Fold increase calculated bydividing the K_(d) of MT4 (6.3 × 10⁻⁹) with the K_(d) of each mutant.³ND = not determined

As evident from Table 6, the K_(d) of the derived Fabs were in the rangeof 10⁻⁹M and each exhibited an increase of gp120 IIIB strain bindingaffinity from 2-3 fold over that of the original Fab MT4. From each ofthe pPho vectors containing the nucleotide sequences encoding the H4H1series Fabs, the nucleotide sequences encoding the histidine-containingheavy chain CDR1 were then separately isolated by a Xho I/Sac II digestand used as described in Example 3 (Table 11) to prepare separatecomposite heavy chains having the preferred CDR1 in combination withaffinity optimized CDR3 prepared as described in C below.

C. Preparation of Randomized CDR3 Contiguous to the PreviouslyRandomized Amino Acids in the Heavy Chain Variable Domain of PhagemidpComb3H-3b3

In order to further enhance the gp120 binding affinity of Fab 3b3 havinga randomized heavy chain CDR1 and CDR3 prepared by overlap PCR asdescribed in Example 1, 15 additional preselected nucleotides in theheavy chain CDR3 of p3b3 subcloned into pComb3H as described in Example1A were subjected to mutagenesis by overlap PCR. To obtain Fab 3b3 asdescribed in Example 1 from pMT4-3, 12 nucleotides encoding four aminoacids, located at the third to the sixth amino acid residue position, inheavy chain CDR3 were randomized. The 15 contiguous nucleotides whichwere 3′ to the first randomized 12 nucleotides were then mutagenized inpComb3H-3b3 to encode five new amino acid residues from the seventh tothe eleventh CDR3 residues as shown in FIGS. 9A and 9B. As a result,Fabs selected from this new library as described below contained a totalof nine randomized amino acid residues of the 18 amino acid residues inthe CDR3 of the Fab 3b3 heavy chain, five newly randomized and selectedamino acids combined with the four previously mutagenized and selectedamino acids in Fab 3b3.

Thus, in essence, since Fab 3b3 was derived originally from pMT4-3, nineamino acid mutations were created by overlap PCR into the heavy chainCDR3 of Fab MT4.

As described in Example 1B, overlap PCR was similarly performed on thepComb3H-3b3 expression vector template previously obtained. In the firstround of PCR, the 5′ FTX3 oligonucleotide primer (SEQ ID NO 9) was usedwith 3′ noncoding mutagenizing oligonucleotide primer, 3b3C35ob, havingthe nucleotide sequence5′-CCAGACGTCCATATAATAATTGTCMNNMNNMNNMNNMNNCCAACCCCACTCCCC CACTCT-3′ (SEQID NO 52) to amplify heavy chain CDR3 and mutagenize the above-described15 nucleotide region. The resultant amplified products were thenpurified as described in Example 1B and used in overlap PCR with theproducts from the second round.

The latter round of PCR was performed also as previously described inExample 1B but with the 5′ coding overlapping oligonucleotide primer,h4h3of, having the nucleotide sequence 5′-GACAATTATTATATGGACGTCTGG-3′(SEQ ID NO 53) and the 3′ noncoding oligonucleotide primer R3B (SEQ IDNO 12). The resultant amplified products were then purified as describedin Example 1B and used in overlap PCR with the products from the firstround using the oligonucleotide primer pair FTX3 and R3B to form apopulation of amplified heavy chain variable domains having newlymutagenized CDR3.

The heavy chain CDR3-randomized amplification products were then ligatedinto pComb3H-TT into which the Sac I/Xba I digested pMT4-3 light chainvariable domain nucleotide sequence (same as p3b3's) was ligated asdescribed in Example 2B to form a library of p3b3-derived pComb3H-CDR3randomized expression vectors. Phage-displayed Fabs, each having amutagenized heavy chain and the pMT4-derived light chain, were thenexpressed and panned on gp120 as described in Example 1.

1) Amino Acid Residue Sequence Analysis of p3b3 Derived Fabs Having aRandomized Heavy Chain CDR3

Clones encoding phage-displayed Fabs that specifically bound to gp120were then sequenced and the derived amino acid residue sequences wereanalyzed as previously described. Nucleic acid sequencing of the CDR3randomized clones produced above was performed on eight randomly chosendouble-stranded Fab-expressing DNA clones using Sequenase 1.0 (USB,Cleveland, Ohio). Alignment of derived amino acid residue sequences withone another and with the Genbank database made use of the MacVectorsuite of programs. The derived heavy chain amino acid residue sequencesof eight selected specific synthetic gp120-specific M556 series Fabs and3b3 are shown in FIGS. 9A and 9B.

All selected M556 Fabs contained both the previously Fab 3b3-mutagenizedand selected CDR1 (NFTVH - SEQ ID NO 3 from residues 28 to 32) and CDR3(EWGW - SEQ ID NO 3 from residues 98-101). Each M556 Fab is unique, asindicated by the numbers following the M556 series designation, byhaving mutagenized and selected amino acid sequences in CDR3corresponding to amino acid residue positions from 102 to 106. Thecomplete heavy chain variable domain amino acid residue sequences of theM556 series are listed in the Sequence Listing as indicated in FIG. 9Bby the assigned Sequence Listing identifiers.

2) Binding Affinity Analysis of gp120-Specific Fabs Having RandomizedHeavy Chain CDR3

The DNA encoding the CDR3-mutagenized heavy chain M556 series Fabsselected for binding to gp120 obtained above were then transferred tothe vector pPho-TT for expression of soluble Fabs as previouslydescribed. The soluble M556 series Fabs were then analyzed for bindingaffinity to gp120 (strain IIIB). Surface plasmon resonance was performedas described in Example 1H to determine the affinity of the selectedFabs.

The results of the Fab binding affinity analysis as measured by theequilibrium dissociation constant (K_(d), M) of the M556 series Fabs arelisted in Table 7. In addition, fold increases of affinity are alsocalculated as previously described.

TABLE 7 ²Fold Increase of Mutant Fab ¹K_(d) K_(d) Compared to MT4 K_(d)M556-2 4.39 × 10⁻¹⁰ 14.3 M556-3 1.01 × 10⁻¹⁰ 63.0 M556-7 1.31 × 10⁻¹⁰48.1 M556-10 9.06 × 10⁻¹¹ 69.5 M556-15 1.74 × 10⁻¹⁰ 36.2 M556-16 1.39 ×10⁻¹⁰ 45.3 M556-5 1.81 × 10⁻⁹  3.5 M556-13 1.01 × 10⁻⁹  6.2¹Dissociation constants of mutants' binding to gp 120 (IIIB strain).²Fold increase calculated by dividing the K_(d) of MT4 (6.3 × 10⁻⁹) withthe K_(d) of each mutant.

As evident from Table 7, six of the eight M556 series Fabs exhibitedenhanced affinities as measured by K_(d) of binding to gp120 IIIB strainin the range of 10⁻¹⁰M to 10⁻¹¹M. Only two, M556-5 and M556-13, hadaffinities in the 10⁻⁹M range. The fold increases of the M556 Fabshaving 10⁻¹⁰M or greater affinities ranged from 14 for M556-2 to 63 forM556-3, and 70 for M556-10, over that of original Fab MT4. Thenucleotide sequences encoding the heavy chain CDR3 from these derivedM556 Fabs were then separately isolated by a Sac II/Spe I digests fromthe pPho vectors and used as described in Example 3 (Table 11) toprepare separate composite heavy chains having the preferred CDR1prepared above in combination with the M556 series affinity optimizedheavy chain CDR3.

D. Preparation of Randomized CDR1 in the Kappa Light Chain VariableDomain of Phagemid pComb3H-3b3 (the pMT4-3 Original Light Chain)

In order to further enhance the gp120 binding affinity of Fab 3b3 havinga randomized heavy chain CDR1 and CDR3 prepared by overlap PCR asdescribed in Example 1, 15 preselected nucleotides in the light chainCDR1 of 3b3 in pComb3H-3b3 were subjected to mutagenesis by overlap PCR.The 3b3 nucleotide sequence having a CDR1 and CDR3 mutagenized heavychain as prepared in Example 1 was subjected to overlap PCR to randomize18 nucleotides from position 76 to 93 in the pComb3H-3b3 light chainvariable domain as shown in FIG. 10 and listed as SEQ ID NO 62. Sincethe p3b3 sequence was derived from pMT4-3 without mutagenizing the lightchain, the reference to the unmutagenized 3b3 light chain in pComb3H isthe same as that to the light chain of pMT4-3. By randomizing theaforementioned 18 nucleotides, the encoded randomized amino acidresidues are in CDR1 from positions 26 to 31 of the complete CDR1 frompositions 22 to 33 as shown on FIGS. 11A and 11B described below.

As a result of the light chain CDR1 mutagenesis of 3b3 in pComb3H-3b3,Fabs selected from this new library as described below contained a totalof six randomized amino acid residues of the 12 amino acid residues inthe Fab 3b3 light chain CDR1 and had the Fab 3b3 CDR1 and CDR3mutagenized and selected heavy chain.

As described in Example 1B, overlap PCR was similarly performed on thepComb3H-3b3 expression vector template previously obtained. In the firstround of PCR, the 5′ oligonucleotide primer, KEF, having the nucleotidesequence 5′-GAATTCTAAACTAGCTAGTCG-3′ (SEQ ID NO 63) was used with 3′noncoding mutagenizing oligonucleotide primer, HIV4cdr1-ov-b, having thenucleotide sequence5′-AGGTTTGTGCTGGTACCAGGCTACMNNMNNMNNMNNMNNMNNGTGACTGGACCTACAGGAGAAGGT-3′ (SEQ ID NO 64) to amplify the light chain CDR1 andmutagenize the above-described 18 nucleotide region. The resultantamplified products were then purified as described in Example 1B andused in overlap PCR with the products from the second round.

The latter round of PCR was performed also as previously described inExample 1B but with the 5′ coding overlapping oligonucleotide primer,HIV4cdr1-fo, having the nucleotide sequence5′-GTAGCCTGGTACCAGCACAAACCT-3′ (SEQ ID NO 65) and the 3′ noncodingoligonucleotide primer, T7B, having the nucleotide sequence5′-AATACGACTCACTATAGGGCG-3′ (SEQ ID NO 66). The resultant amplifiedproducts were then purified as described in Example 1B and used inoverlap PCR with the products from the first round using theoligonucleotide primer pair KEF and T7B to form a population ofamplified light chain variable domains having newly mutagenized CDR1.

The light chain CDR1-randomized amplification products were then ligatedinto pComb3H-TT into which the p3b3 heavy chain variable domainnucleotide sequence encoding the amino acid residue sequence shown inSEQ ID NO 3 was ligated as described in Example 2B to form a library ofp3b3-derived pComb3H-CDR1 light chain randomized expression vectors.Phage-displayed Fabs, each having a mutagenized light chain and thep3b3-derived heavy chain, were then expressed and panned on gp120 asdescribed in Example 1.

1) Amino Acid Residue Sequence Analysis of T3b3 Derived Fabs Having aRandomized Light Chain CDR1

Clones encoding phage-displayed Fabs that specifically bound to gp120were then sequenced and the derived amino acid residue sequences wereanalyzed as previously described. Nucleic acid sequencing of the CDR1randomized clones produced above was performed on four randomly chosendouble-stranded Fab-expressing DNA clones using Sequenase 1.0 (USE,Cleveland, Ohio). The derived light chain amino acid residue sequencesof the four selected specific synthetic gp120-specific A-D series ofFabs and 3b3 are shown in FIG. 11.

All selected A-D Fabs contained the p3b3-derived heavy chain variabledomain. Each A-D Fab is unique by having mutagenized and selected aminoacid sequences in the light chain CDR1 corresponding to amino acidresidue positions from 26 to 31, within the complete CDR1 from aminoacid residue positions 22 to 33 as listed in SEQ ID NOs 67-70. Thecomplete light chain variable domain amino acid residue sequences of theA-D series are listed in the Sequence Listing as indicated in FIG. 11 bythe assigned Sequence Listing identifiers.

2) Binding Affinity Analysis of gp120-Specific Fabs Having RandomizedLight Chain CDR1

The DNA encoding the CDR1 mutagenized light chain A-D series Fabsselected for binding to gp120 obtained above were then transferred tothe vector pPho-TT for expression of soluble Fabs as previouslydescribed. The soluble A-D series Fabs were then analyzed for bindingaffinity to gp120 strain IIIB. Surface plasmon resonance was performedas described in Example 1H to determine the affinity of the selectedFabs.

The results of the Fab binding affinity analysis as measured by theequilibrium dissociation constant (K_(d), M) of the A-D series Fabs arelisted in Table 8. In addition, fold increases of affinity are alsocalculated as previously described.

TABLE 8 ²Fold Increase of Mutant Fab ¹K_(d) K_(d) Compared to MT4 K_(d)A 4.7 × 10⁻⁹  1.30 B 1.1 × 10⁻⁹  5.70 C 6.8 × 10⁻¹⁰ 9.26 D 2.2 × 10⁻¹⁰28.60 ¹Dissociation constants of mutants' binding to gp 120 (IIIBstrain). ²Fold increase calculated by dividing the K_(d) of MT4 (6.3 ×10⁻⁹) with the K_(d) of each mutant.

As evident from Table 8, two Fabs, Fab C and Fab D, of the four A-Dseries Fabs exhibited enhanced affinities as measured by K_(d) ofbinding to gp120 IIIB strain in the range of 10⁻¹⁰M. Fabs A and B hadaffinities in the 10⁻⁹M range. The fold increases of Fab C and Fab Dwere respectively approximately 9 and 28 fold over that of original FabMT4 from which Fab 3b3 was derived. The fold increase as compared to theFab 3b3 template from which Fabs A-D were derived can also be similarlycalculated by using the K_(d) of Fab 3b3 which is 7.7×10⁻¹⁰M.

The nucleotide sequences encoding the light chain variable domains fromthese derived CDR1-mutagenized Fabs were then separately used astemplates for further mutagenesis procedures as described in Example 3.In addition, the nucleotide sequences encoding the light chain CDR1 ofthe Fabs A-D obtained herein were separately isolated by a Sac I/Kpn Idigests from the pPho vectors and used as described in Example 3 (Table11) to prepare separate composite light chains having the preferred CDR1prepared above in combination with the H4L3 series affinity optimizedlight chain CDR3 prepared below.

E. Preparation of Randomized CDR3 in the Kappa Light Chain VariableDomain of Phagemid DComb3H-3b3 (the DMT4-3 Original Light Chain)

In order to further enhance the gp120 binding affinity of Fab 3b3 havinga randomized heavy chain CDR1 and CDR3 prepared by overlap PCR asdescribed in Example 1, 15 discontinuous preselected nucleotides in thelight chain CDR3 of 3b3 in pComb3H were subjected to mutagenesis byoverlap PCR. The 3b3 nucleotide sequences isolated from p3b3 having aCDR1 and CDR3 mutagenized heavy chain as prepared in Example 1 weresubjected to overlap PCR to randomize 15 nucleotides from positions 265to 267 and then from 271 to 282, the sites thus separated by oneunmutagenized triplet in the p3b3-derived light chain variable domain asshown in FIG. 10 and listed as SEQ ID NO 62. Since p3b3 was derived frompMT4-3 without mutagenizing the light chain, the reference to theunmutagenized p3b3-derived light chain is the same as that to the lightchain of pMT4-3. By randomizing the aforementioned 15 nucleotides, theencoded randomized amino acid residues are in CDR3 at position 89 thenfrom positions 91 to 94 leaving conserved glutamine and tyrosineresidues respectively in positions 88 and 90 and conserved tyrosine andthreonine residues respectively in positions 95 and 96 therebycomprising the nine amino acid light chain CDR3.

As a result of the light chain CDR3 mutagenesis of 3b3 in pComb3H-3b3,Fabs selected from this new library as described below contained a totalof five randomized amino acid residues of the nine amino acid residuesin the Fab 3b3 light chain CDR3 and had the Fab 3b3 CDR1 and CDR3mutagenized and selected heavy chain.

As described in Example 1B, overlap PCR was similarly performed on thepComb3H-3b3 expression vector template previously obtained. In the firstround of PCR, the 5′ oligonucleotide primer, KEF (SEQ ID NO 63) was usedwith 3′ noncoding mutagenizing oligonucleotide primer, h4kcdr3-bo,having the nucleotide sequence5′-CAGTTTGGTCCCCTGGCCAAAAGTGTAMNNMNNMNNMNNATAMNNCTGACAGTAGTACAGTGCAAAGTC-3′ (SEQ ID NO 71) to amplify the light chain CDR3 andmutagenize the above-described 15 nucleotide region. The resultantamplified products were then purified as described in Example 1B andused in overlap PCR with the products from the second round.

The latter round of PCR was performed also as previously described inExample 1B but with the 5′ coding overlapping oligonucleotide primer,hvkfr4-fo, having the nucleotide sequence5′-TACACTTTTGGCCAGGGGACCAAACTG-3′ (SEQ ID NO 72) and the 3′ noncodingoligonucleotide primer, T7B (SEQ ID NO 66). The resultant amplifiedproducts were then purified as described in Example 1B and used inoverlap PCR with the products from the first round using theoligonucleotide primer pair KEF and T7B to form a population ofamplified light chain variable domains having newly mutagenized CDR3.

The light chain CDR3-randomized amplification products were then ligatedinto pComb3H-TT into which the p3b3 heavy chain variable domainnucleotide sequence encoding the amino acid residue sequence shown inSEQ ID NO 3 was ligated as described in Example 2B to form a library ofp3b3-derived pComb3H-CDR3 randomized expression vectors. Phage-displayedFabs, each having a mutagenized light chain and the p3b3-derived heavychain, were then expressed and panned on gp120 as described in Example1.

1) Amino Acid Residue Sequence Analysis of p3b3 Derived Fabs Having aRandomized Light Chain CDR3

Clones encoding phage-displayed Fabs that specifically bound to gp120were then sequenced and the derived amino acid residue sequences wereanalyzed as previously described. Nucleic acid sequencing of the CDR3randomized clones produced above was performed on three randomly chosendouble-stranded Fab-expressing DNA clones using Sequenase 1.0 (USB,Cleveland, Ohio). The derived light chain amino acid residue sequencesof the three selected specific synthetic gp120-specific H4L3 series ofFabs and 3b3 are shown in FIGS. 12A and 12B.

All selected H4L3 Fabs contained the p3b3-derived heavy chain variabledomain. Each H4L3 Fab is unique by having mutagenized and selected aminoacid sequences in CDR3 corresponding to the positions noted above. Thecomplete light chain variable domain amino acid residue sequences of theH4L3 series are listed in the Sequence Listing as indicated in FIG. 12Bby the assigned Sequence Listing identifiers.

2) Binding Affinity Analysis of gp120-Specific Fabs Having RandomizedLight Chain CDR3

The DNA encoding the CDR3-mutagenized light chain H4L3 series Fabsselected for binding to gp120 obtained above were then transferred tothe vector pPho-TT for expression of soluble Fabs as previouslydescribed. The soluble H4L3 series Fabs were then analyzed for bindingaffinity to gp120 strain IIIB. Surface plasmon resonance was performedas described in Example 1H to determine the affinity of the selectedFabs.

The results of the Fab binding affinity analysis as measured by theequilibrium dissociation constant (K_(d), M) of the H4L3 series Fabs arelisted in Table 9. In addition, fold increases of affinity are alsocalculated as previously described.

TABLE 9 ²Fold Increase of Mutant Fab ¹K_(d) K_(d) Compared to MT4 K_(d)H4L3-2 1.38 × 10⁻¹⁰ 45.3 H4L3-3 3.31 × 10⁻⁹  1.9 H4L3-4 2.43 × 10⁻¹⁰25.7 ¹Dissociation constants of mutants' binding to gp 120 (IIIBstrain). ²Fold increase calculated by dividing the K_(d) of MT4 (6.3 ×10⁻⁹) with the K_(d) of each mutant.

As evident from Table 9, two of the three Fabs, H4L3-2 and H4L3-4,exhibited enhanced affinities as measured by K_(d) of binding to gp120IIIB strain in the range of 10⁻¹⁰M corresponding respectively to foldincreases of approximately 45 and 26 fold over that of original Fab MT4from which Fab 3b3, and thus the A-D Fabs, was derived. The foldincrease as compared to the Fab 3b3 template from which Fabs A-D werederived can also be similarly calculated by using the K_(d) of Fab 3b3which is 7.7×10⁻¹⁰M.

The nucleotide sequences encoding the light chain variable domains fromthese derived CDR3 mutagenized Fabs were then separately used to createcomposite light chain variable domains having optimized mutagenized andselected CDR. Specifically, the nucleotide sequences encoding themutagenized light chain CDR3 were isolated by Kpn I/Xba I digestion fromthe H4L3-pPho vectors and used as described in Example 3 (Table 11) toprepare separate composite light chains having the preferred CDR3prepared above in combination with the A-D series affinity optimizedlight chain CDR1 prepared above.

3. Preparation of Randomized CDR Composite Fabs Having OptimizedAffinity to gp120 Based Upon Preselected Randomized CDR of Phagemids 3b3and MT4

A. Preparation of Randomized CDR3 Light Chain Based on Phagemid D Havinga Randomized CDR1

1) PCR Amplification

In order to create a light chain having both randomized CDR1 and CDR3,one of the phagemids prepared in Example 2D, phagemid D, having arandomized CDR1 was used as a template for the subsequent randomizationof CDR3. As described in Examples 1 and 2, the mutagenized light chainof phagemid D was derived from the nonrandomized light chain of 3b3,which is also referred to as the original light chain MT4 or 4L. In 3b3,the nonrandomized light chain of MT4 was retained while the heavy chainwas randomized in CDR1 and CDR3. Since phagemid D was derived fromphagemid 3b3 in pComb3H-3b3, phagemid D thus has the previouslyrandomized 3b3-derived heavy chain and a newly randomized MT4 or 3b3light chain in CDR1.

In this Example, phagemid D having a randomized CDR1 and CDR3 heavychain sequence and a randomized CDR1 light chain sequence was subjectedto light chain CDR3 mutagenesis as described herein. A preselectedportion of the CDR3 light chain of phagemid D was selected forrandomization by overlap PCR as described in Example 1B. The CDR3originally from MT4 as described above contained 27 nucleotides thatexpressed the nine amino acid residue sequence QVYGASSYT (SEQ ID NO 6,from amino acid residue position 88 to 96). In mutagenizing the CDR3light chain, the second (valine), and fourth through seventh amino acidresidues (glycine, alanine, serine, serine) were randomized as dictatedby the design of the oligonucleotide primer used to mutagenize theregion. The first, third, eighth and ninth residues of the light chainCDR3 were retained as they are conserved residues found in light chains.

In the first round of PCR amplification, the phagemid D template wasmutagenized using the 5′ oligonucleotide primer KEF (SEQ ID NO 63)described in Example 2D and the 3′ noncoding randomizing oligonucleotideprimer h4kcdr3-bo (SEQ ID NO 71). The PCR products were purified aspreviously described and used in overlap PCR with the products of thesecond PCR.

The latter was performed on phagemid D with the 5′ codingoligonucleotide primer hvkfr4-fo (SEQ ID NO 72) and the 3′ noncodingoligonucleotide primer T7B (SEQ ID NO 66) described in Example 2D.

The PCR products of the second amplification were purified as previouslydescribed and used in overlap PCR with the products of the first PCR inoverlap PCR with the primer pair KEF and T7B which provided for theoverlap between the 5′ end of the coding hvkfr4fo primer and the 5═ endof the noncoding randomizing h4kcdr3bo primer. The library of overlapPCR products of the phagemid D-derived light chain having a populationof randomized CDR3 in the context of the remainder of the phagemid Dsequence, already having randomized and selected light chain CDR1 andheavy chain CDR1 and CDR3, were then purified as described in Example 1Band ligated into pComb3H-TT as described in Example 2B for subsequentphage-display Fab expression and screening as described below.

2) Selection of Anti-gp120 Fabs Having CDR1 and CDRB Randomized LightChains

The construction of pComb3H-phagemid D-derived and newly mutagenizedlight chain CDR3-containing libraries was performed as previouslydescribed in Example 2B with the exception that the heavy chain variabledomain cassette was obtained from phagemid D and light chain variabledomain was provided in the amplified fragments having mutagenized CDR3.Following expression and panning as previously described, gp120-reactivephage-displayed Fabs were subjected to acid elution as previouslydescribed. The clones encoding the Fabs designated QA1 through QA9 wereobtained using this standard elution procedure. When the affinities ofthe corresponding soluble Fabs were determined as described below and asshown in Table 10, the affinities were not as enhanced by themutagenesis procedure as anticipated.

A separate elution procedure was, therefore, used to obtain FabD-derived Fabs having higher affinities for gp120. In this procedure,the washed gp120-bound phage-displayed Fabs were competed by incubating10 ug/well of soluble Fab 3b3 in a total volume of 25 ul for 1 to 2hours. By competing with excess Fab in this way, the low affinitybinders were eluted leaving the higher affinity binders immobilized withthe gp120 for subsequent elution with acid performed as previouslydescribed. The clones obtained through the Fab competition/acid elutionprocedure encoded the Fabs designated QA10, QA11 and QA12, as shown inFIGS. 13A and 13B and in Table 10 below.

3) Amino Acid Residue Sequence Analysis Comparison Between the ParentFab D and the Fab D-based CDR1 and CDR3 Light Chain Randomized Fabs

Clones encoding phagemid D-derived Fabs prepared above that specificallybound to gp120 and recovered using the elution procedures describedabove were then sequenced and the derived amino acid residue sequenceswere analyzed. Nucleic acid sequencing of the CDR3 randomized clonesproduced above was performed on four randomly chosen double-strandedFab-expressing DNA clones using Sequenase 1.0 (USB, Cleveland, Ohio).The derived light chain amino acid residue sequences of the 12 selectedspecific synthetic gp120-specific QA series of Fabs and 3b3 are shown inFIGS. 13A and 13B.

All selected QA series Fabs contained the phagemid D-derived heavy chainvariable domain, which is the p3b3 heavy chain as described in Example2D, and the previously mutagenized and selected light chain CDR1 havingthe amino acid residue sequence RSSHQLDGSRVA (SEQ ID NO 70 from residuepositions 22 to 33). Each QA Fab is unique by having mutagenized andselected amino acid sequences in the light chain CDR3 corresponding tothe positions noted above. The complete light chain variable domainamino acid residue sequences of the QA series are listed in the SequenceListing as indicated in FIG. 13B by the assigned Sequence Listingidentifiers. The nucleotide sequence and corresponding encoded lightchain variable domain from the Fab designated QA11 is referred to as LHfor preparation of composite Fabs as described below.

4) Binding Affinity Analysis of gp120-Specific Fabs Derived fromPhagemid D Having CDR1 and CDR3 Randomized Light Chain Domains

The DNA encoding the CDR3-mutagenized light chain QA series Fabsselected for binding to gp120 obtained above were then transferred tothe vector pPho-TT for expression of soluble Fabs as previouslydescribed. The soluble QA series Fabs were then analyzed for bindingaffinity to gp120 strain IIIB. Surface plasmon resonance was performedas described in Example 1H to determine the affinity of the selectedFabs.

The results of the Fab binding affinity analysis as measured by theequilibrium dissociation constant (K_(d), M) of the QA11 series Fabs arelisted in Table 10. In addition, fold increases of affinity are alsocalculated as previously described.

TABLE 10 ²Fold Increase of Mutant Fab ¹K_(d) K_(d) Compared to MT4 K_(d)QA1 5.1 × 10⁻¹⁰ 12.2 QA2 4.2 × 10⁻¹⁰ 14.9 QA3 1.2 × 10⁻⁹  5.1 QA4 5.0 ×10⁻¹⁰ 12.6 QA5 4.6 × 10⁻¹⁰ 13.7 QA6 2.2 × 10⁻¹⁰ 28.6 QA7 4.5 × 10⁻⁹ 1.43 QA8 4.4 × 10⁻¹⁰ 14.3 QA9 6.1 × 10⁻¹⁰ 10.3  QA10 7.4 × 10⁻¹¹ 85.9 QA11 6.7 × 10⁻¹¹ 94.4  QA12 1.3 × 10⁻¹⁰ 48.6 ¹Dissociation constants ofmutants' binding to gp 120 (IIIB strain). ²Fold increase calculated bydividing the K_(d) of MT4 (6.3 × 10⁻⁹) with the K_(d) of each mutant.

As shown in Table 10, the Fab-expressing phage-displayed clones obtainedfrom the Fab competition/acid elution exhibited higher affinities forgp120 as expected. While most of the QA Fabs had K_(d)'s greater than10⁻¹⁰M, the QA10 and QA11 Fabs had K_(d)'s exceeding 10⁻¹⁰M of bindingto gp120 IIIB strain with the latter corresponding respectively to foldincreases of approximately 86 and 94 fold over that of original Fab MT4from which Fab 3b3 and Fab D were derived.

The nucleotide sequences encoding the light chain variable domains fromthese derived CDR3 mutagenized Fabs were then separately used increating unique composite affinity-optimized Fabs having a new heavy andlight chain variable domains as described below and shown in Table 12and 13.

B. Preparation of Composite Fabs by Combining reselected Heavy and LightChain Constructs

1) Preparation of pPho Constructs for Expressing Either Composite Heavyor Light Chains

The nucleotide sequences encoding gp120-specific high affinity Fabsproduced by the CDR-directed mutagenesis approach as taught in thisinvention and in Examples 1, 2 and 3A were then used to create new heavyand light chain variable domain constructs. The latter were then used toexpress new gp120 affinity-optimized heavy and light chain variabledomains as components of new Fab compositions of this invention asdescribed in Example 3B2) below.

In Table 11, four new heavy chain (V_(H)) phagemid constructs and onenew light chain (V_(K)) phagemid construct were prepared using from thepPho-based constructs that contain nucleotide sequences encoding thespecifically identified Fabs. To form the new heavy or light chaincomposite constructs, double restriction digests of selected pPho-Fabexpressing constructs were performed resulting in isolating DNAfragments in which mutagenized CDR were contained. For all of the heavychain digests, Xho I cuts in framework I, Spe I cuts in framework 4 andSac II cuts in framework II at nucleotide positions 203 to 208 as shownin FIG. 4 and listed in SEQ ID NO 7. For all of the light chain digests,Sac I cuts in framework I, Xba I cuts in framework 4 and Kpn I cuts inframework II at nucleotide positions 100 to 105 as shown in FIG. 10 andlisted in SEQ ID NO 62.

The particular digested fragments to form each composite heavy or lightchain are identified in Table 11 along with the respective SEQ ID NO foreach amino acid residue sequence encoded by the composite nucleotidesequence. In addition, the composite amino acid residue sequence for thelight chain composite, designated L42, is shown in FIGS. 14A and 14B andthe composite amino acid residue sequence for the heavy chaincomposites, designated H31, H33, H101 and H103, are shown in FIGS. 15Aand 15B.

TABLE 11 Phagemid DNA Chain Fragments Ligated to V_(H) or V_(K) ChainSEQ ID Form V_(H) or V_(K) Chain Composite¹ NO. Composite Sequence H31(V_(H)) 89 H4H1-1 (Xho I/Sac II) + M556-3 (Sac II/Spe I) H33 (V_(H)) 90H4H1-3 (Xho I/Sac II) + M556-3 (Sac II/Spe I) H101 (V_(H)) 91 H4H1-1(Xho I/Sac II) + M556-10 (Sac II/Spe I) H103 (V_(H)) 92 H4H1-3 (XhoI/Sac II) + M556-10 (Sac II/Spe I) L42 (V_(K)) 88 D (Sac I/Kpn I) +H4L3-2 (Kpn I/Xba I) ¹Amino acid residue sequences of V_(H) or V_(K)(kappa light chain) chain composites are shown in FIGS. 15 and 14,respectively, and listed in the Sequence Listing according to thedesignated SEQ ID NOs.

In order to express the heavy and light chain composites, the isolatedfragments were first ligated with the complementary termini resultingfrom the digestion to form one heavy or light chain fragment. The latterwere then separately ligated with a similarly digested pPho-TT vectorfor directional ligation of the fragments into the vector for subsequentexpression thereof for analysis of binding affinity as described below.Moreover, with the clones encoding the optimized Fabs produced inExamples 1, 2 and 3A, additional heavy and light chain composites aresimilarly prepared by ligating preselected isolated heavy or light chainDNA fragments.

2) Preparation of pPho Constructs for Expressing Unique Fabs HavingRandomized CDR by Combining Preselected Heavy and Light Chain Constructs

Using the heavy and light chain composite DNA constructs prepared abovealong with the nucleotide sequence encoding the original pMT4-derivedlight chain, designated 4L, the nucleotide sequence encoding theQA11-derived light chain (also referred to as LH), and the nucleotidesequence encoding the pMT4 original heavy chain designated 4H, uniqueDNA constructs for encoding preselected pairs of heavy and light chainswere prepared.

The new Fab-encoding pPho constructs were prepared by combining XhoI/Spe I heavy chain constructs with Sac I/Xba I light chain constructsin pPho-TT by serial ligations. Alternatively, the pPho constructs inwhich either a preselected heavy or light chain construct was alreadypresent were digested with restriction enzymes for ligating the otherchain as dictated by the chain present in the vector. For example, theexpression vector, pPho-MT4, having the heavy and light chain constructsfor expressing MT4, was digested with Sac I/Xba I for directionalligation of the L42 composite light chain construct prepared above.Similar constructions are accomplished by methods well known to one ofordinary skill in the art.

Preferred optimized composite constructs for encoding preferred uniquehigh affinity binding Fabs of this invention are listed in Table 12. Thepairs of heavy and light chain amino acid residue sequences of eachpreferred composite Fab are also indicated in Table 12 as pairs of SEQID NOs separated by a colon, e.g., 89:6 for the composite Fab having themutagenized composite heavy chain H31 paired with the originalunmutagenized light chain 4L.

TABLE 12 Optimized Composite Heavy (V_(H)):Light (V_(K)) FabDesignations Chain SEQ ID NOs H31/4L 89:6 H31/L42 89:88 4H/L42  1:88H103/L42 92:88 H33/L42 90:88 H33/LH (LH = QA11 V_(K)) 90:86 H101/L4291:88 H101/4L (4L = MT4 V_(K)) 91:6 

Other pairs of heavy and light chains in addition to those listed inTable 12 are similarly constructed for expression of additional uniquecomposite Fabs that exhibit binding to gp120.

3) Binding Affinity Analysis of Optimally CDR Randomized Composite FabsHaving Enhanced Affinity for gp120

For determination of the enhancement of affinity accomplished with theheavy and light chain CDR-directed mutagenesis optimization proceduresas described in these Examples, soluble forms of the selected compositeFabs were expressed from the pPho composite constructs prepared above.The soluble composite Fabs were then analyzed for binding affinity togp120 strain IIIB. Surface plasmon resonance was performed as describedin Example 1H to determine the affinity of the selected composite Fabs.

The results of the composite Fab binding affinity analysis as measuredby the association rate constants, K_(on), and K_(off), are shown inTable 13 and the calculated equilibrium association and dissociationconstants (K_(a), M⁻¹, K_(d), M) are listed in Table 14. Selectedindividual clones are indicated by numbers separated by a dash from theFab designation. In addition, fold increases of affinity are alsocalculated as previously described for each of the composite Fabscompared to that of the unmutagenized and nonoptimized gp120-binding MT4Fab.

TABLE 13 Fab K_(on) K_(off) H31/ 1.53 × 10⁵ ± 2.6 × 10⁴ 3.39 × 10⁻⁶ ±1.11 × 10⁻⁷ 4L-14 H31/ 6.73 × 10⁴ ± 6.5 × 10³ 1.75 × 10⁻⁷ ± 1.5 × 10⁻⁷L42-1 4H*/ 2.96 × 10⁵ ± 1.8 × 10⁴ 6.15 × 10⁻⁴ ± 1.92 × 10⁻⁶ L42-1 H103/8.18 × 10⁴ ± 6.5 × 10³ 9.41 × 10⁻⁶ ± 1.15 × 10⁻⁷ L42-4 H33/ 9.84 × 10⁴ ±6.5 × 10³ 1.78 × 10⁻⁵ ± 1.45 × 10⁻⁷ L42-17 H33/ 7.32 × 10⁴ ±   1 × 10⁴5.45 × 10⁻⁶ ± 1.08 × 10⁻⁷ LH-11 H101/ 6.91 × 10⁴ ± 7.9 × 10³ 1.19 × 10⁻⁵± 9.65 × 10⁻⁸ L42-16 H101/ 1.34 × 10⁵ ± 8.7 × 10³ 1.74 × 10⁻⁵ ± 1.12 ×10⁻⁷ L42-1 H101/ 8.93 × 10⁵ ± 1.3 × 10⁴ 3.39 × 10⁻⁵ ± 2.58 × 10⁻⁷ 4L-1*4H = Heavy Cha1n from MT4

TABLE 14 ¹Fold Increase of Mutant K_(d) Compared Fab K_(a) K_(d) to MT4K_(d) H31/ 4.51 × 10¹⁰ 2.22 × 10⁻¹¹ 283.8 4L-14 H31/ 3.85 × 10¹¹ 2.59 ×10⁻¹² 2432.4 L42-1 24H/ 4.81 × 10⁸ 2.08 × 10⁻⁹ 3.02 L42-1 H103/ 8.88 ×10⁹ 1.13 × 10⁻¹⁰ 55.8 L42-4 H33/ 5.46 × 10⁹ 1.83 × 10⁻¹⁰ 34.4 L42-17H33/ 1.36 × 10¹⁰ 7.38 × 10⁻¹¹ 85.4 LH-11 H101/ 5.81 × 10⁹ 1.72 × 10⁻¹⁰36.6 L42-16 H101/  7.7 × 10⁹ 1.30 × 10⁻¹⁰ 48.5 L42-1 H101/ 2.63 × 10⁹3.80 × 10⁻¹⁰ 16.6 4L-1 ¹Fold increase calculated by dividing the K_(d)of MT4 (6.3 × 10⁻⁹) with the K_(d) of each mutant optimized compositeFab ²4H = Heavy chain from MT4

As shown in the above Tables, the Fab-expressing phage-displayed clonesobtained from selectively combining mutagenesis-optimized heavy andlight chain variable domain DNA cassettes encoded Fabs exhibiting highaffinity binding to gp120. In particular, the Fabs in which either heavyor light chain CDR were mutagenized and selected for optimal gp120binding exhibited K_(d)'s greater than 10⁻¹⁰M, with two Fabs havingK_(d)'s exceeding 10⁻¹¹M, and one preferred Fab designated H31/L42-1exhibiting a K_(d) greater than 10⁻¹²M. The corresponding fold increasesof gp120 binding affinity ranged from approximately 16 to over 2400 foldover that of original Fab MT4. In addition, the half-life of the bindingof the gp120-reactive Fabs to gp120 can be obtained by dividing thenatural log (ln₂) with the K_(off) rate constant, thereby obtaining afurther parameter of the nature of the interaction. For the Fabdesignated H31/L42-l, the calculated half-life of binding to gp120 is44.5 days compared to 24 minutes for the nonrandomized and nonoptimizedFab MT4 derived from a HIV-seropositive asymptomatic patient. Thus, thehalf-life determination is another way of characterizing the highaffinity binding of the synthetic optimized Fabs of this invention togp120 of HIV.

The Fab designated H31/L42-1 having the highest affinity was a Fabcomposite of the heavy chain composite H31 comprising heavy chainCDR1-mutagenized Fab H4H1 and heavy chain CDR3-mutagenized Fab MS56-3added in combination with the light chain composite L42 comprising lightchain CDR1-mutagenized region from Fab D and light chainCDR3-mutagenized Fab H4L3-2.

A cell culture of E. coli strain DH10B containing the pPho construct inwhich the nucleotide sequence encoding the Fab H31/L42-1 is present hasbeen deposited with American Type Culture Collection on Sep. 16, 1994,as described in Example 4 and has been assigned the ATCC AccessionNumber 69691.

Thus, the methods of this invention, in utilizing CDR-directedmutagenesis of both heavy and light chain genes, has generated uniqueFabs not previously available by evolutionary selection pressures norpresent in a patient generating endogenous antibodies directed againstgp120 of HIV. The composite Fabs prepared in Example 3 along with thegp120-reactive Fabs selected from the four libraries prepared in Example2 represent Fabs exhibiting higher affinity binding to gp120 than anyother currently known gp120-reactive Fabs or intact immunoglobulins.

The preferred synthetic Fabs exhibiting high affinity binding to gp120as obtained and characterized as described herein are listed in Table 15where each Fab designation is listed along with the correspondingaffinity measurement in K_(d), M. In addition, the amino acid residuesequences of the heavy and light chain variable domains comprising thepreferred Fabs of this invention are indicated in Table 15 by pairs ofSEQ ID NOs separated by a colon. For example, the heavy and light chainamino acid residue sequences of the highest affinity binding Fab,H31/L42-1, respectively correspond to SEQ ID NO 89 and 88 and are listedas such in the Sequence Listing.

TABLE 15 SEQ ID NOs of Fab Designation K_(d) V_(H):V_(K) Pairs MT4 (=Fab 4) 6.3 × 10⁻⁹ 1:6 3b3 7.7 × 10⁻¹⁰ 3:6 M556-2 4.39 × 10⁻¹⁰ 54:6M556-3 1.0 × 10⁻¹⁰ 55:6 M556-7 1.3 × 10⁻¹⁰ 56:6 M556-10 9.06 × 10⁻¹⁰57:6 M556-15 1.74 × 10⁻¹⁰ 58:6 M556-16 1.39 × 10⁻¹⁰ 59:6 C 6.8 × 10⁻¹⁰3:69 D 2.2 × 10⁻¹⁰ 3:70 H4L3-2 1.38 × 10⁻¹⁰ 3:73 H4L3-4 2.43 × 10⁻¹⁰3:75 QA1 5.1 × 10⁻¹⁰ 3:76 QA2 4.2 × 10⁻¹⁰ 3:77 QA4 5.0 × 10⁻¹⁰ 3:79 QA54.6 × 10⁻¹⁰ 3:80 QA6 2.2 × 10⁻¹⁰ 3:82 QA8 4.4 × 10⁻¹⁰ 3:83 QA9 6.1 ×10⁻¹⁰ 3:84 QA10 7.4 × 10⁻¹¹ 3:85 QA11 6.7 × 10⁻¹¹ 3:86 QA12 1.3 × 10⁻¹⁰3:87 ¹H31/4L-14 2.2 × 10⁻¹¹ 89:6 ²H31/L42-1 2.59 × 10⁻¹² 89:88H103/L42-4 1.13 × 10⁻¹⁰ 92:88 H33/L42-17 1.83 × 10⁻¹⁰ 90:88 ³H33/LH-117.38 × 10⁻¹¹ 90:86 ⁴H101/L42-16 1.7 × 10⁻¹⁰ 91:88 ⁴H101/L42-1 1.3 ×10⁻¹⁰ 91:88 H101/4L-1 3.8 × 10⁻¹⁰ 91:6 ¹4L = MT4 or 3b3 V_(K) ²Fabexpressed by plasmid pPho-H31/L42-1 in E. coli ATCC deposit 69691 ³LH =light chain of QA11 ⁴Separate selected Fabs having the same V_(H)/V_(K)

4. Deposit of Materials

The plasmid, pMT4, was deposited on Oct. 19, 1993, with the AmericanType Culture Collection, 1301 Parklawn Drive, Rockville, Md., USA (ATCC)and has been assigned the ATCC accession number 75574. The depositprovides a plasmid that encodes and expresses a Fab antibody designatedMT4, the heavy and light chain variable domain amino acid sequences ofwhich are listed in SEQ ID NO 1 and 6, respectively, and are shown inFIGS. 1 and 2.

The cell culture, designated DH10B-pPho-H31/L42-1, containing plasmidpPho-H31/L42-1, was deposited on or before Sep. 19, 1994 with ATCC andhas been assigned the ATCC accession number 69691. The deposit providesan E. coli cell culture containing a pPho-based plasmid from which a Fabantibody designated H31/L42-1 is expressed, the heavy and light chainamino acid sequences of which are listed in SEQ ID NO 89 and 88,respectively, and are shown in FIGS. 15 and 14.

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable plasmid and viable cellculture for 30 years from the date of each deposit. The plasmid and cellculture will be made available by ATCC under the terms of the BudapestTreaty which assures permanent and unrestricted availability of theprogeny of the plasmid and culture to the public upon issuance of thepertinent U.S. patent or upon laying open to the public of any U.S. orforeign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 U.S.C.§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14 with particular reference to 886 OG 638). The assignee of thepresent application has agreed that if the plasmid and culture depositsshould die or be lost or destroyed when cultivated under suitableconditions, it will be promptly replaced on notification with a viablespecimen of the same plasmid and culture. Availability of the depositsis not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell lines deposited,since the deposited embodiments are intended as single illustrations ofone aspect of the invention and any cell lines that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial does not constitute an admission that the written descriptionherein contained is inadequate to enable the practice of any aspect ofthe invention, including the best mode thereof, nor is it to beconstrued as limiting the scope of the claims to the specificillustration that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

92 124 amino acids amino acid linear protein not provided 1 Leu Glu GlnSer Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val SerCys Gln Ala Ser Gly Tyr Arg Phe Ser Asn Phe Val Ile His 20 25 30 Trp ValArg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn ProTyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val ThrPhe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 ArgSer Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 GlyPro Tyr Ser Trp Asp Asp Ser Pro Gln Asp Asn Tyr Tyr Met Asp 100 105 110Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 124 amino acidsamino acid linear protein not provided 2 Leu Glu Gln Ser Gly Ala Glu ValLys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala Ser GlyTyr Arg Phe Ser Asn Phe Thr Leu Met 20 25 30 Trp Val Arg Gln Ala Pro GlyGln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn LysGlu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala Asp ThrSer Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg Ser AlaAsp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly Gln Trp Asn Trp AspAsp Ser Pro Gln Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys GlyThr Thr Val Ile Val Ser Ser 115 120 124 amino acids amino acid linearprotein not provided 3 Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro GlyAla Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe SerAsn Phe Thr Val His 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln Arg Phe GluTrp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser AlaLys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser Ala Asn ThrAla Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp Thr Ala ValTyr Tyr Cys Ala Arg Val 85 90 95 Gly Glu Trp Gly Trp Asp Asp Ser Pro GlnAsp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Thr Val IleVal Ser Ser 115 120 123 amino acids amino acid linear protein notprovided 4 Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser ValLys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser Asn Tyr ThrLeu Ile 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met GlyTrp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe GlnAsp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr MetGlu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr CysAla Arg Val 85 90 95 Gly Pro Trp Asn Trp Asp Ser Pro Gln Asp Asn Tyr TyrMet Asp Val 100 105 110 Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115120 124 amino acids amino acid linear protein not provided 5 Leu Glu GlnSer Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val SerCys Gln Ala Ser Gly Tyr Arg Phe Ser Asn Phe Thr Val His 20 25 30 Trp ValArg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn ProTyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val ThrPhe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 ArgSer Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 GlyPro Trp Arg Trp Asp Asp Ser Pro Gln Asp Asn Tyr Tyr Met Asp 100 105 110Val Trp Gly Lys Gly Thr Ile Val Ile Val Ser Ser 115 120 108 amino acidsamino acid linear protein not provided 6 Glu Leu Thr Gln Ser Pro Gly ThrLeu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys Arg SerSer His Ser Ile Arg Ser Arg Arg Val 20 25 30 Ala Trp Tyr Gln His Lys ProGly Gln Ala Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg Ala SerGly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe ThrLeu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu Tyr TyrCys Gln Val Tyr Gly Ala Ser Ser Tyr Thr 85 90 95 Phe Gly Gln Gly Thr LysLeu Glu Arg Lys Arg Thr 100 105 372 base pairs nucleic acid singlelinear cDNA not provided 7 CTCGAGCAGT CTGGGGCTGA GGTGAAGAAG CCTGGGGCCTCAGTGAAGGT TTCTTGTCAA 60 GCTTCTGGAT ACAGATTCAG TAACTTTGTT ATTCATTGGGTGCGCCAGGC CCCCGGACAG 120 AGGTTTGAGT GGATGGGATG GATCAATCCT TACAACGGAAACAAAGAATT TTCAGCGAAG 180 TTCCAGGACA GAGTCACCTT TACCGCGGAC ACATCCGCGAACACAGCCTA CATGGAGTGG 240 AGGAGCCTCA GATCTGCAGA CACGGCTGTT TATTATTGTGCGAGAGTGGG GCCATATAGT 300 TGGGATGATT CTCCCCAGGA CAATTATTAT ATGGACGTCTGGGGCAAAGG GACCACGGTC 360 ATCGTCTCCT CA 372 5 amino acids amino acidlinear protein not provided 8 Asn Phe Val Ile His 1 5 22 base pairsnucleic acid single linear DNA (genomic) not provided 9 GCAATTAACCCTCACTAAAG GG 22 25 base pairs nucleic acid single linear DNA (genomic)not provided 10 AGAAGCTTGA CAAGAAGAAA CCTTC 25 70 base pairs nucleicacid single linear DNA (genomic) not provided 11 GAAGGTTTCT TGTCAAGCTTCTGGATACAG ATTCAGTNNS NNSNNSNNSN NSTGGGTGCG 60 CCAGGCCCCC 70 21 basepairs nucleic acid single linear DNA (genomic) not provided 12TTGATATTCA CAAACGAATG G 21 75 base pairs nucleic acid single linear DNA(genomic) not provided 13 CCCTTTGCCC CAGACGTCCA TATAATAATT GTCCTGGGGAGAATCATCMN NMNNMNNMNN 60 CCCCACTCTC GCACA 75 5 amino acids amino acidlinear protein not provided 14 Arg Tyr Thr Val Phe 1 5 5 amino acidsamino acid linear protein not provided 15 Asn Trp Ser Val Met 1 5 5amino acids amino acid linear protein not provided 16 Gly Tyr Thr LeuMet 1 5 5 amino acids amino acid linear protein not provided 17 Asn PheThr Leu Leu 1 5 5 amino acids amino acid linear protein not provided 18His Tyr Ser Leu Met 1 5 5 amino acids amino acid linear protein notprovided 19 Asn Trp Val Val His 1 5 5 amino acids amino acid linearprotein not provided 20 Asn Phe Ser Ile Met 1 5 5 amino acids amino acidlinear protein not provided 21 Asn Phe Ala Ile His 1 5 5 amino acidsamino acid linear protein not provided 22 Asn Phe Thr Met Val 1 5 5amino acids amino acid linear protein not provided 23 Asn Phe Thr LeuGln 1 5 5 amino acids amino acid linear protein not provided 24 Tyr PheThr Met His 1 5 5 amino acids amino acid linear protein not provided 25Ser Tyr Pro Leu His 1 5 5 amino acids amino acid linear protein notprovided 26 Asn Phe Thr Leu Met 1 5 5 amino acids amino acid linearprotein not provided 27 Asn Tyr Thr Ile Met 1 5 5 amino acids amino acidlinear protein not provided 28 Asn Phe Thr Val His 1 5 5 amino acidsamino acid linear protein not provided 29 Asn Tyr Thr Leu Ile 1 5 5amino acids amino acid linear protein not provided 30 Asn Phe Ile IleMet 1 5 5 amino acids amino acid linear protein not provided 31 Asn PheSer Ile Met 1 5 5 amino acids amino acid linear protein not provided 32Asn Tyr Thr Ile Gln 1 5 5 amino acids amino acid linear protein notprovided 33 Asn Phe Thr Val His 1 5 4 amino acids amino acid linearprotein not provided 34 Pro Tyr Ser Trp 1 4 amino acids amino acidlinear protein not provided 35 Gln Trp Asn Trp 1 4 amino acids aminoacid linear protein not provided 36 Pro Trp Thr Trp 1 4 amino acidsamino acid linear protein not provided 37 Glu Trp Gly Trp 1 4 aminoacids amino acid linear protein not provided 38 Pro Trp Asn Trp 1 4amino acids amino acid linear protein not provided 39 Leu Trp Asn Trp 14 amino acids amino acid linear protein not provided 40 Ser Trp Arg Trp1 4 amino acids amino acid linear protein not provided 41 Pro Tyr SerTrp 1 4 amino acids amino acid linear protein not provided 42 Pro TrpArg Trp 1 4691 base pairs nucleic acid single circular DNA (genomic) notprovided 43 GGGAAATTGT AAGCGTTAAT ATTTTGTTAA AATTCGCGTT AAATTTTTGTTAAATCAGCT 60 CATTTTTTAA CCAATAGGCC GAAATCGGCA AAATCCCTTA TAAATCAAAAGAATAGACCG 120 AGATAGGGTT GAGTGTTGTT CCAGTTTGGA ACAAGAGTCC ACTATTAAAGAACGTGGACT 180 CCAACGTCAA AGGGCGAAAA ACCGTCTATC AGGGCGATGG CCCACTACGTGAACCATCAC 240 CCTAATCAAG TTTTTTGGGG TCGAGGTGCC GTAAAGCACT AAATCGGAACCCTAAAGGGA 300 GCCCCCGATT TAGAGCTTGA CGGGGAAAGC CGGCGAACGT GGCGAGAAAGGAAGGGAAGA 360 AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG CAAGTGTAGC GGTCACGCTGCGCGTAACCA 420 CCACACCCGC CGCGCTTAAT GCGCCGCTAC AGGGCGCGTC AGGTGGCACTTTTCGGGGAA 480 ATGTGCGCGG AACCCCTATT TGTTTATTTT TCTAAATACA TTCAAATATGTATCCGCTCA 540 TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA AAGGAAGAGTATGAGTATTC 600 AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCTGTTTTTGCTC 660 ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCACGAGTGGGTT 720 ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCCGAAGAACGTT 780 TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCCCGTATTGACG 840 CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTGGTTGAGTACT 900 CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTATGCAGTGCTG 960 CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATCGGAGGACCGA 1020 AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTTGATCGTTGGG 1080 AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATGCCTGTAGCAA 1140 TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCTTCCCGGCAAC 1200 AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGCTCGGCCCTTC 1260 CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCTCGCGGTATCA 1320 TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTACACGACGGGGA 1380 GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCCTCACTGATTA 1440 AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT TTAGATTGATTTAAAACTTC 1500 ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA TAATCTCATGACCAAAATCC 1560 CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT AGAAAAGATCAAAGGATCTT 1620 CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA AACAAAAAAACCACCGCTAC 1680 CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAGGTAACTGGCT 1740 TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTAGGCCACCACT 1800 TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT AATCCTGTTACCAGTGGCTG 1860 CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC AAGACGATAGTTACCGGATA 1920 AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA GCCCAGCTTGGAGCGAACGA 1980 CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA AAGCGCCACGCTTCCCGAAG 2040 GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAGCGCACGAGGG 2100 AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT CGGGTTTCGCCACCTCTGAC 2160 TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG CCTATGGAAAAACGCCAGCA 2220 ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT TGCTCACATGTTCTTTCCTG 2280 CGTTATCCCC TGATTCTGTG GATAACCGTA TTACCGCCTT TGAGTGAGCTGATACCGCTC 2340 GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA GGAAGCGGAAGAGCGCCCAA 2400 TACGCAAACC GCCTCTCCCC GCGCGTTGGC CGATTCATTA ATGCAGCTGGCACGACAGGT 2460 TTCCCGACTG GAAAGCGGGC AGTGAGCGCA ACGCAATTAA TGTGAGTTAGCTCACTCATT 2520 AGGCACCCCA GGCTTTACAC TTTATGCTTC CGGCTCGTAT GTTGTGTGGAATTGTGAGCG 2580 GATAACAATT GAATTCAGGA GGAATTTAAA ATGAAAAAGA CAGCTATCGCGATTGCAGTG 2640 GCACTGGCTG GTTTCGCTAC CGTGGCCCAG GCGGCCGAGC TCACGCAGTCTCCAGGCACC 2700 CTGTCTTTGT CTCCAGGGGA AAGAGCCACC CTCTCCTGCA GGGCCAGTCACAGTGTTAGC 2760 AGGGCCTACT TAGCCTGGTA CCAGCAGAAA CCTGGCCAGG CTCCCAGGCTCCTCATCTAT 2820 GGTACATCCA GCAGGGCCAC TGGCATCCCA GACAGGTTCA GTGGCAGTGGGTCTGGGACA 2880 GACTTCACTC TCACCATCAG CAGACTGGAG CCTGAAGATT TTGCAGTGTACTACTGTCAG 2940 CAGTATGGTG GCTCACCGTG GTTCGGCCAA GGGACCAAGG TGGAACTCAAACGAACTGTG 3000 GCTGCACCAT CTGTCTTCAT CTTCCCGCCA TCTGATGAGC AGTTGAAATCTGGAACTGCC 3060 TCTGTTGTGT GCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACAGTGGAAGGTG 3120 GATAACGCCC TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGACAGCAAGGAC 3180 AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG CAGACTACGAGAAACACAAA 3240 GTCTACGCCT GCGAAGTCAC CCATCAGGGC CTGAGTTCGC CCGTCACAAAGAGCTTCAAC 3300 AGGGGAGAGT GTTAATTCTA GATAATTAAT TAGGAGGAAT TTAAAATGAAATACCTATTG 3360 CCTACGGCAG CCGCTGGATT GTTATTACTC GCTGCCCAAC CAGCCATGGCCGAGGTGCAG 3420 CTGCTCGAGC AGTCTGGGGC TGAGGTGAAG AAGCCTGGGT CCTCGGTGAAGGTCTCCTGC 3480 AGGGCTTCTG GAGGCACCTT CAACAATTAT GCCATCAGCT GGGTGCGACAGGCCCCTGGA 3540 CAAGGGCTTG AGTGGATGGG AGGGATCTTC CCTTTCCGTA ATACAGCAAAGTACGCACAA 3600 CACTTCCAGG GCAGAGTCAC CATTACCGCG GACGAATCCA CGGGCACAGCCTACATGGAG 3660 CTGAGCAGCC TGAGATCTGA GGACACGGCC ATATATTATT GTGCGAGAGGGGATACGATT 3720 TTTGGAGTGA CCATGGGATA CTACGCTATG GACGTCTGGG GCCAAGGGACCACGGTCACC 3780 GTCTCCGCAG CCTCCACCAA GGGCCCATCG GTCTTCCCCC TGGCACCCTCCTCCAAGAGC 3840 ACCTCTGGGG GCACAGCGGC CCTGGGCTGC CTGGTCAAGG ACTACTTCCCCGAACCGGTG 3900 ACGGTGTCGT GGAACTCAGG CGCCCTGACC AGCGGCGTGC ACACCTTCCCGGCTGTCCTA 3960 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAGCAGCTTGGGC 4020 ACCCAGACCT ACATCTGCAA CGTGAATCAC AAGCCCAGCA ACACCAAGGTGGACAAGAAA 4080 GCAGAGCCCA AATCTTGTGA CAAAACTAGT GGCCAGGCCG GCCAGGAGGGTGGTGGCTCT 4140 GAGGGTGGCG GTTCTGAGGG TGGCGGCTCT GAGGGAGGCG GTTCCGGTGGTGGCTCTGGT 4200 TCCGGTGATT TTGATTATGA AAAGATGGCA AACGCTAATA AGGGGGCTATGACCGAAAAT 4260 GCCGATGAAA ACGCGCTACA GTCTGACGCT AAAGGCAAAC TTGATTCTGTCGCTACTGAT 4320 TACGGTGCTG CTATCGATGG TTTCATTGGT GACGTTTCCG GCCTTGCTAATGGTAATGGT 4380 GCTACTGGTG ATTTTGCTGG CTCTAATTCC CAAATGGCTC AAGTCGGTGACGGTGATAAT 4440 TCACCTTTAA TGAATAATTT CCGTCAATAT TTACCTTCCC TCCCTCAATCGGTTGAATGT 4500 CGCCCTTTTG TCTTTAGCGC TGGTAAACCA TATGAATTTT CTATTGATTGTGACAAAATA 4560 AACTTATTCC GTGGTGTCTT TGCGTTTCTT TTATATGTTG CCACCTTTATGTATGTATTT 4620 TCTACGTTTG CTAACATACT GCGTAATAAG GAGTCTTAAG CTAGCTAATTAATTTAAGCG 4680 GCCGCAGATC T 4691 6 amino acids amino acid linearpeptide not provided 44 Glu Val Gln Leu Leu Glu 1 5 124 amino acidsamino acid linear protein not provided 45 Leu Glu Gln Ser Gly Ala GluVal Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala SerGly Tyr Arg Phe Ser His Phe Thr Val His 20 25 30 Trp Val Arg Gln Ala ProGly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly AsnLys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala AspThr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg SerAla Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly Pro Tyr Ser TrpAsp Asp Ser Pro Gln Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly LysGly Thr Thr Val Ile Val Ser Ser 115 120 124 amino acids amino acidlinear protein not provided 46 Leu Glu Gln Ser Gly Ala Glu Val Lys LysPro Gly Ala Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr ArgPhe Ser His Phe Thr Leu His 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln ArgPhe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu PheSer Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser AlaAsn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp ThrAla Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly Pro Tyr Ser Trp Asp Asp SerPro Gln Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr ThrVal Ile Val Ser Ser 115 120 124 amino acids amino acid linear proteinnot provided 47 Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala SerVal Lys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser His PheThr Ile Met 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp MetGly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys PheGln Asp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala TyrMet Glu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr TyrCys Ala Arg Val 85 90 95 Gly Pro Tyr Ser Trp Asp Asp Ser Pro Gln Asp AsnTyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Thr Val Ile Val SerSer 115 120 124 amino acids amino acid linear protein not provided 48Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 1015 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser Asn Tyr Thr Leu Gln 20 2530 Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 4045 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 5560 Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 7075 80 Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 8590 95 Gly Pro Tyr Ser Trp Asp Asp Ser Pro Gln Asp Asn Tyr Tyr Met Asp100 105 110 Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 124amino acids amino acid linear protein not provided 49 Leu Glu Gln SerGly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser CysGln Ala Ser Gly Tyr Arg Phe Ser Asn Phe Thr Leu Ile 20 25 30 Trp Val ArgGln Ala Pro Gly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro TyrAsn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr PheThr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg SerLeu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly ProTyr Ser Trp Asp Asp Ser Pro Gln Asp Asn Tyr Tyr Met Asp 100 105 110 ValTrp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 124 amino acidsamino acid linear protein not provided 50 Leu Glu Gln Ser Gly Ala GluVal Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala SerGly Tyr Arg Phe Ser Asn Trp Thr Ile Met 20 25 30 Trp Val Arg Gln Ala ProGly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly AsnLys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala AspThr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg SerAla Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly Pro Tyr Ser TrpAsp Asp Ser Pro Gln Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly LysGly Thr Thr Val Ile Val Ser Ser 115 120 6166 base pairs nucleic acidsingle circular DNA (genomic) not provided 51 TAATGCGGTA GTTTATCACAGTTAAATTGC TAACGCAGTC AGGCACCGTG TATGAAATCT 60 AACAATGCGC TCATCGTCATCCTCGGCACC GTCACCCTGG ATGCTGTAGG CATAGGCTTG 120 GTTATGCCGG TACTGCCGGGCCTCTTGCGG GATATCGTCC ATTCCGACAG CATCGCCAGT 180 CACTATGGCG TGCTGCTAGCGCTATATGCG TTGATGCAAT TTCTATGCGC ACCCGTTCTC 240 GGAGCACTGT CCGACCGCTTTGGCCGCCGC CCAGTCCTGC TCGCTTCGCT ACTTGGAGCC 300 ACTATCGACT ACGCGATCATGGCGACCACA CCCGTCCTGT GGATCCTCTA CGCCGGACGC 360 ATCGTGGCCG GCATCACCGGCGCCACAGGT GCGGTTGCTG GCGCCTATAT CGCCGACATC 420 ACCGATGGGG AAGATCGGGCTCGCCACTTC GGGCTCATGA GCGCTTGTTT CGGCGTGGGT 480 ATGGTGGCAG GCCCCGTGGCCGGGGGACTG TTGGGCGCCA TCTCCTTGCA TGCACCATTC 540 CTTGCGGCGG CGGTGCTCAACGGCCTCAAC CTACTACTGG GCTGCTTCCT AATGCAGGAG 600 TCGCATAAGG GAGAGCGTCGACCGATGCCC TTGAGAGCCT TCAACCCAGT CAGCTCCTTC 660 CGGTGGGCGC GGGGCATGACTATCGTCGCC GCACTTATGA CTGTCTTCTT TATCATGCAA 720 CTCGTAGGAC AGGTGCCGGCAGCGCTCTGG GTCATTTTCG GCGAGGACCG CTTTCGCTGG 780 AGCGCGACGA TGATCGGCCTGTCGCTTGCG GTATTCGGAA TCTTGCACGC CCTCGCTCAA 840 GCCTTCGTCA CTGGTCCCGCCACCAAACGT TTCGGCGAGA AGCAGGCCAT TATCGCCGGC 900 ATGGCGGCCG ACGCGCTGGGCTACGTCTTG CTGGCGTTCG CGACGCGAGG CTGGATGGCC 960 TTCCCCATTA TGATTCTTCTCGCTTCCGGC GGCATCGGGA TGCCCGCGTT GCAGGCCATG 1020 CTGTCCAGGC AGGTAGATGACGACCATCAG GGACAGCTTC AAGGATCGCT CGCGGCTCTT 1080 ACCAGCCTAA CTTCGATCACTGGACCGCTG ATCGTCACGG CGATTTATGC CGCCTCGGCG 1140 AGCACATGGA ACGGGTTGGCATGGATTGTA GGCGCCGCCC TATACCTTGT CTGCCTCCCC 1200 GCGTTGCGTC GCGGTGCATGGAGCCGGGCC ACCTCGACCT GAATGGAAGC CGGCGGCACC 1260 TCGCTAACGG ATTCACCACTCCAAGAATTG GAGCCAATCA ATTCTTGCGG AGAACTGTGA 1320 ATGCGCAAAC CAACCCTTGGCAGAACATAT CCATCGCGTC CGCCATCTCC AGCAGCCGCA 1380 CGCGGCGCAT CTCGGGCAGCGTTGGGTCCT GGCCACGGGT GCGCATGATC GTGCTCCTGT 1440 CGTTGAGGAC CCGGCTAGGCTGGCGGGGTT GCCTTACTGG TTAGCAGAAT GAATCACCGA 1500 TACGCGAGCG AACGTGAAGCGACTGCTGCT GCAAAACGTC TGCGACCTGA GCAACAACAT 1560 GAATGGTCTT CGGTTTCCGTGTTTCGTAAA GTCTGGAAAC GCGGAAGTCA GCGCCCTGCA 1620 CCATTATGTT CCGGATCTGCATCGCAGGAT GCTGCTGGCT ACCCTGTGGA ACACCTACAT 1680 CTGTATTAAC GAAGCGCTGGCATTGACCCT GAGTGATTTT TCTCTGGTCC CGCCGCATCC 1740 ATACCGCCAG TTGTTTACCCTCACAACGTT CCAGTAACCG GGCATGTTCA TCATCAGTAA 1800 CCCGTATCGT GAGCATCCTCTCTCGTTTCA TCGGTATCAT TACCCCCATG AACAGAAATT 1860 CCCCCTTACA CGGAGGCATCAAGTGACCAA ACAGGAAAAA ACCGCCCTTA ACATGGCCCG 1920 CTTTATCAGA AGCCAGACATTAACGCTTCT GGAGAAACTC AACGAGCTGG ACGCGGATGA 1980 ACAGGCAGAC ATCTGTGAATCGCTTCACGA CCACGCTGAT GAGCTTTACC GCAGCTGCCT 2040 CGCGCGTTTC GGTGATGACGGTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC 2100 AGCTTGTCTG TAAGCGGATGCCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT 2160 TGGCGGGTGT CGGGGCGCAGCCATGACCCA GTCACGTAGC GATAGCGGAG TGTATACTGG 2220 CTTAACTATG CGGCATCAGAGCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA 2280 CCGCACAGAT GCGTAAGGAGAAAATACCGC ATCAGGCGCT CTTCCGCTTC CTCGCTCACT 2340 GACTCGCTGC GCTCGGTCGTTCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA 2400 ATACGGTTAT CCACAGAATCAGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG 2460 CAAAAGGCCA GGAACCGTAAAAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC 2520 CCTGACGAGC ATCACAAAAATCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA 2580 TAAAGATACC AGGCGTTTCCCCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG 2640 CCGCTTACCG GATACCTGTCCGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCATAGC 2700 TCACGCTGTA GGTATCTCAGTTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC 2760 GAACCCCCCG TTCAGCCCGACCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC 2820 CCGGTAAGAC ACGACTTATCGCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG 2880 AGGTATGTAG GCGGTGCTACAGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA 2940 AGGACAGTAT TTGGTATCTGCGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT 3000 AGCTCTTGAT CCGGCAAACAAACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG 3060 CAGATTACGC GCAGAAAAAAAGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT 3120 GACGCTCAGT GGAACGAAAACTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG 3180 ATCTTCACCT AGATCCTTTTAAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT 3240 GAGTAAACTT GGTCTGACAGTTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC 3300 TGTCTATTTC GTTCATCCATAGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG 3360 GAGGGCTTAC CATCTGGCCCCAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT 3420 CCAGATTTAT CAGCAATAAACCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA 3480 ACTTTATCCG CCTCCATCCAGTCTATTAAT TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG 3540 CCAGTTAATA GTTTGCGCAACGTTGTTGCC ATTGCTGCAG GCATCGTGGT GTCACGCTCG 3600 TCGTTTGGTA TGGCTTCATTCAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC 3660 CCCATGTTGT GCAAAAAAGCGGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG 3720 TTGGCCGCAG TGTTATCACTCATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG 3780 CCATCCGTAA GATGCTTTTCTGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG 3840 TGTATGCGGC GACCGAGTTGCTCTTGCCCG GCGTCAACAC GGGATAATAC CGCGCCACAT 3900 AGCAGAACTT TAAAAGTGCTCATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG 3960 ATCTTACCGC TGTTGAGATCCAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA 4020 GCATCTTTTA CTTTCACCAGCGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA 4080 AAAAAGGGAA TAAGGGCGACACGGAAATGT TGAATACTCA TACTCTTCCT TTTTCAATAT 4140 TATTGAAGCA TTTATCAGGGTTATTGTCTC ATGAGCGGAT ACATATTTGA ATGTATTTAG 4200 AAAAATAAAC AAATAGGGGTTCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTCCTG 4260 CAGTGGAGAT TATCGTCACTGCAATGCTTC GCAATATGGC GCAAAATGAC CAACAGCGGT 4320 TGATTGATCA GGTAGAGGGGGCGCTGTACG AGGTAAAGCC CGATGCCAGC ATTCCTGACG 4380 ACGATACGGA GCTGCTGCGCGATTACGTAA AGAAGTTATT GAAGCATCCT CGTCAGTAAA 4440 AAGTTAATCT TTTCAACAGCTGTCATAAAG TTGTCACGGC CGAGACTTAT AGTCGCTTTG 4500 TTTTTATTTT TTAATGTATTGAATTCAGGA GGAATTTAAA ATGAAAAAGA CAGCTATCGC 4560 GATTGCAGTG GCACTGGCTGGTTTCGCTAC CGTGGCCCAG GCGGCCGAGC TCACGCAGTC 4620 TCCAGGCACC CTGTCTTTGTCTCCAGGGGA AAGAGCCACC CTCTCCTGCA GGGCCAGTCA 4680 CAGTGTTAGC AGGGCCTACTTAGCCTGGTA CCAGCAGAAA CCTGGCCAGG CTCCCAGGCT 4740 CCTCATCTAT GGTACATCCAGCAGGGCCAC TGGCATCCCA GACAGGTTCA GTGGCAGTGG 4800 GTCTGGGACA GACTTCACTCTCACCATCAG CAGACTGGAG CCTGAAGATT TTGCAGTGTA 4860 CTACTGTCAG CAGTATGGTGGCTCACCGTG GTTCGGCCAA GGGACCAAGG TGGAACTCAA 4920 ACGAACTGTG GCTGCACCATCTGTCTTCAT CTTCCCGCCA TCTGATGAGC AGTTGAAATC 4980 TGGAACTGCC TCTGTTGTGTGCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACA 5040 GTGGAAGGTG GATAACGCCCTCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA 5100 CAGCAAGGAC AGCACCTACAGCCTCAGCAG CACCCTGACG CTGAGCAAAG CAGACTACGA 5160 GAAACACAAA GTCTACGCCTGCGAAGTCAC CCATCAGGGC CTGAGTTCGC CCGTCACAAA 5220 GAGCTTCAAC AGGGGAGAGTGTTAATTCTA GATAATTAAT TAGGAGGAAT TTAAAATGAA 5280 ATACCTATTG CCTACGGCAGCCGCTGGATT GTTATTACTC GCTGCCCAAC CAGCCATGGC 5340 CGAGGTGCAG CTGCTCGAGCAGTCTGGGGC TGAGGTGAAG AAGCCTGGGT CCTCGGTGAA 5400 GGTCTCCTGC AGGGCTTCTGGAGGCACCTT CAACAATTAT GCCATCAGCT GGGTGCGACA 5460 GGCCCCTGGA CAAGGGCTTGAGTGGATGGG AGGGATCTTC CCTTTCCGTA ATACAGCAAA 5520 GTACGCACAA CACTTCCAGGGCAGAGTCAC CATTACCGCG GACGAATCCA CGGGCACAGC 5580 CTACATGGAG CTGAGCAGCCTGAGATCTGA GGACACGGCC ATATATTATT GTGCGAGAGG 5640 GGATACGATT TTTGGAGTGACCATGGGATA CTACGCTATG GACGTCTGGG GCCAAGGGAC 5700 CACGGTCACC GTCTCCGCAGCCTCCACCAA GGGCCCATCG GTCTTCCCCC TGGCACCCTC 5760 CTCCAAGAGC ACCTCTGGGGGCACAGCGGC CCTGGGCTGC CTGGTCAAGG ACTACTTCCC 5820 CGAACCGGTG ACGGTGTCGTGGAACTCAGG CGCCCTGACC AGCGGCGTGC ACACCTTCCC 5880 GGCTGTCCTA CAGTCCTCAGGACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG 5940 CAGCTTGGGC ACCCAGACCTACATCTGCAA CGTGAATCAC AAGCCCAGCA ACACCAAGGT 6000 GGACAAGAAA GCAGAGCCCAAATCTTGTGA CAAAACTAGT GGCCAGGCCG GCCAGTAATT 6060 AATTAGCCCG CCTAATGAGCGGGCTTTTTT TTAAGCGGCC GCTTATCATC GATAAGCTTT 6120 CGTCTTCAAG ATTTCTCATGTTTGACAGCT TATCATCGAT AAGCTT 6166 60 base pairs nucleic acid singlelinear DNA (genomic) not provided 52 CCAGACGTCC ATATAATAAT TGTCMNNMNNMNNMNNMNNC CAACCCCACT CCCCCACTCT 60 24 base pairs nucleic acid singlelinear DNA (genomic) not provided 53 GACAATTATT ATATGGACGT CTGG 24 124amino acids amino acid linear protein not provided 54 Leu Glu Gln SerGly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser CysGln Ala Ser Gly Tyr Arg Phe Ser Asn Phe Thr Val His 20 25 30 Trp Val ArgGln Ala Pro Gly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro TyrAsn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr PheThr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg SerLeu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly GluTrp Gly Trp Glu Gln Phe Arg Phe Asp Asn Tyr Tyr Met Asp 100 105 110 ValTrp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 124 amino acidsamino acid linear protein not provided 55 Leu Glu Gln Ser Gly Ala GluVal Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala SerGly Tyr Arg Phe Ser Asn Phe Thr Val His 20 25 30 Trp Val Arg Gln Ala ProGly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly AsnLys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala AspThr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg SerAla Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly Glu Trp Gly TrpGlu Met Phe Arg Tyr Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly LysGly Thr Thr Val Ile Val Ser Ser 115 120 124 amino acids amino acidlinear protein not provided 56 Leu Glu Gln Ser Gly Ala Glu Val Lys LysPro Gly Ala Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr ArgPhe Ser Asn Phe Thr Val His 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln ArgPhe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu PheSer Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser AlaAsn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp ThrAla Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly Glu Trp Gly Trp Glu Met ArgArg Phe Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr ThrVal Ile Val Ser Ser 115 120 124 amino acids amino acid linear proteinnot provided 57 Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala SerVal Lys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser Asn PheThr Val His 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp MetGly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys PheGln Asp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala TyrMet Glu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr TyrCys Ala Arg Val 85 90 95 Gly Glu Trp Gly Trp His Gln Arg Arg Tyr Asp AsnTyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Thr Val Ile Val SerSer 115 120 124 amino acids amino acid linear protein not provided 58Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 1015 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser Asn Phe Thr Val His 20 2530 Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 4045 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 5560 Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 7075 80 Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 8590 95 Gly Glu Trp Gly Trp Thr Gln Arg Arg Phe Asp Asn Tyr Tyr Met Asp100 105 110 Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 124amino acids amino acid linear protein not provided 59 Leu Glu Gln SerGly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser CysGln Ala Ser Gly Tyr Arg Phe Ser Asn Phe Thr Val His 20 25 30 Trp Val ArgGln Ala Pro Gly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro TyrAsn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr PheThr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg SerLeu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly GluTrp Gly Trp Asp Gln Val Arg Tyr Asp Asn Tyr Tyr Met Asp 100 105 110 ValTrp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 124 amino acidsamino acid linear protein not provided 60 Leu Glu Gln Ser Gly Ala GluVal Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala SerGly Tyr Arg Phe Ser Asn Phe Thr Val His 20 25 30 Trp Val Arg Gln Ala ProGly Gln Arg Phe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly AsnLys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala AspThr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg SerAla Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly Glu Trp Gly TrpAsp Gln Arg Arg Tyr Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly LysGly Thr Thr Val Ile Val Ser Ser 115 120 124 amino acids amino acidlinear protein not provided 61 Leu Glu Gln Ser Gly Ala Glu Val Lys LysPro Gly Ala Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr ArgPhe Ser Asn Phe Thr Val His 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln ArgPhe Glu Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu PheSer Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser AlaAsn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp ThrAla Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly Glu Trp Gly Trp Glu Met AlaIle Gln Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr ThrVal Ile Val Ser Ser 115 120 324 base pairs nucleic acid single linearDNA (genomic) not provided 62 GAGCTCACGC AGTCTCCAGG CACCCTGTCTCTGTCTCCAG GGGAAAGAGC CACCTTCTCC 60 TGTAGGTCCA GTCACAGCAT TCGCAGCCGCCGCGTAGCCT GGTACCAGCA CAAACCTGGC 120 CAGGCTCCAA GGCTGGTCAT ACATGGTGTTTCCAATAGGG CCTCTGGCAT CTCAGACAGG 180 TTCAGCGGCA GTGGGTCTGG GACAGACTTCACTCTCACCA TCACCAGAGT GGAGCCTGAA 240 GACTTTGCAC TGTACTACTG TCAGGTCTATGGTGCCTCCT CGTACACTTT TGGCCAGGGG 300 ACCAAACTGG AGAGGAAACG AACT 324 21base pairs nucleic acid single linear DNA (genomic) not provided 63GAATTCTAAA CTAGCTAGTC G 21 66 base pairs nucleic acid single linear DNA(genomic) not provided 64 AGGTTTGTGC TGGTACCAGG CTACMNNMNN MNNMNNMNNMNNGTGACTGG ACCTACAGGA 60 GAAGGT 66 24 base pairs nucleic acid singlelinear DNA (genomic) not provided 65 GTAGCCTGGT ACCAGCACAA ACCT 24 21base pairs nucleic acid single linear DNA (genomic) not provided 66AATACGACTC ACTATAGGGC G 21 108 amino acids amino acid linear protein notprovided 67 Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly GluArg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser Ser His Lys Glu Phe Gly ArgArg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu ValIle His 35 40 45 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val GluPro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr Cys Gln Val Tyr Gly Ala SerSer Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100105 108 amino acids amino acid linear protein not provided 68 Glu LeuThr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 AlaThr Phe Ser Cys Arg Ser Ser His Thr Val Tyr Arg Asp Arg Val 20 25 30 AlaTrp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 GlyVal Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 GlySer Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80Asp Phe Ala Leu Tyr Tyr Cys Gln Val Tyr Gly Ala Ser Ser Tyr Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100 105 108 amino acidsamino acid linear protein not provided 69 Glu Leu Thr Gln Ser Pro GlyThr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys ArgSer Ser His Pro Leu His Arg Ala Arg Val 20 25 30 Ala Trp Tyr Gln His LysPro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg AlaSer Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp PheThr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu TyrTyr Cys Gln Gln Tyr Gly Trp Pro Phe Tyr Thr 85 90 95 Phe Gly Gln Gly ThrLys Leu Glu Arg Lys Arg Thr 100 105 108 amino acids amino acid linearprotein not provided 70 Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu SerPro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser Ser His Gln LeuAsp Gly Ser Arg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro Gly Gln Ala ProArg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser AspArg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile ThrArg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr Cys Gln Val TyrGly Ala Ser Ser Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Arg LysArg Thr 100 105 69 base pairs nucleic acid single linear DNA (genomic)not provided 71 CAGTTTGGTC CCCTGGCCAA AAGTGTAMNN MNNMNNMNNA TAMNNCTGACAGTAGTACAG 60 TGCAAAGTC 69 27 base pairs nucleic acid single linear DNA(genomic) not provided 72 TACACTTTTG GCCAGGGGAC CAAACTG 27 108 aminoacids amino acid linear protein not provided 73 Glu Leu Thr Gln Ser ProGly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser CysArg Ser Ser His Ser Ile Arg Ser Arg Arg Val 20 25 30 Ala Trp Tyr Gln HisLys Pro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn ArgAla Ser Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala LeuTyr Tyr Cys Gln Gln Tyr Gly Trp Pro Phe Tyr Thr 85 90 95 Phe Gly Gln GlyThr Lys Leu Glu Arg Lys Arg Thr 100 105 108 amino acids amino acidlinear protein not provided 74 Glu Leu Thr Gln Ser Pro Gly Thr Leu SerLeu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser Ser HisSer Ile Arg Ser Arg Arg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro Gly GlnAla Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg Ala Ser Gly IleSer Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr Cys GlnVal Tyr Gly Gly Ser Ala Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu GluArg Lys Arg Thr 100 105 108 amino acids amino acid linear protein notprovided 75 Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly GluArg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser Ser His Ser Ile Arg Ser ArgArg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu ValIle His 35 40 45 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val GluPro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr Cys Gln Lys Tyr Gly Gly GlyThr Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100105 108 amino acids amino acid linear protein not provided 76 Glu LeuThr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 AlaThr Phe Ser Cys Arg Ser Ser His Gln Leu Asp Gly Ser Arg Val 20 25 30 AlaTrp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 GlyVal Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 GlySer Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80Asp Phe Ala Leu Tyr Tyr Cys Gln Val Tyr Gly Trp Ser Gln Tyr Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100 105 108 amino acidsamino acid linear protein not provided 77 Glu Leu Thr Gln Ser Pro GlyThr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys ArgSer Ser His Gln Leu Asp Gly Ser Arg Val 20 25 30 Ala Trp Tyr Gln His LysPro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg AlaSer Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp PheThr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu TyrTyr Cys Gln Leu Tyr Gly Arg Gly Asn Tyr Thr 85 90 95 Phe Gly Gln Gly ThrLys Leu Glu Arg Lys Arg Thr 100 105 108 amino acids amino acid linearprotein not provided 78 Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu SerPro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser Ser His Gln LeuAsp Gly Ser Arg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro Gly Gln Ala ProArg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser AspArg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile ThrArg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr Cys Gln Thr TyrGly Arg Gly Val Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Arg LysArg Thr 100 105 108 amino acids amino acid linear protein not provided79 Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 510 15 Ala Thr Phe Ser Cys Arg Ser Ser His Gln Leu Asp Gly Ser Arg Val 2025 30 Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val Ile His 3540 45 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser Gly Ser 5055 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 6570 75 80 Asp Phe Ala Leu Tyr Tyr Cys Gln Ser Tyr Gly Gly Arg Asp Tyr Thr85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100 105 108amino acids amino acid linear protein not provided 80 Glu Leu Thr GlnSer Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr PheSer Cys Arg Ser Ser His Gln Leu Asp Gly Ser Arg Val 20 25 30 Ala Trp TyrGln His Lys Pro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 Gly Val SerAsn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser GlyThr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80 Asp PheAla Leu Tyr Tyr Cys Gln Thr Tyr Gly Trp Ser Gly Tyr Thr 85 90 95 Phe GlyGln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100 105 108 amino acids aminoacid linear protein not provided 81 Glu Leu Thr Gln Ser Pro Gly Thr LeuSer Leu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser SerHis Gln Leu Asp Gly Ser Arg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro GlyGln Ala Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg Ala Ser GlyIle Ser Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr CysGln Lys Tyr Gly Asp Ser Phe Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys LeuGlu Arg Lys Arg Thr 100 105 108 amino acids amino acid linear proteinnot provided 82 Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro GlyGlu Arg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser Ser His Gln Leu Asp GlySer Arg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg LeuVal Ile His 35 40 45 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg PheSer Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg ValGlu Pro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr Cys Gln Met Tyr Gly GlyArg Asp Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr100 105 108 amino acids amino acid linear protein not provided 83 GluLeu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15Ala Thr Phe Ser Cys Arg Ser Ser His Gln Leu Asp Gly Ser Arg Val 20 25 30Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 7580 Asp Phe Ala Leu Tyr Tyr Cys Gln Gln Tyr Gly Asp Ser Leu Tyr Thr 85 9095 Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100 105 108 aminoacids amino acid linear protein not provided 84 Glu Leu Thr Gln Ser ProGly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser CysArg Ser Ser His Gln Leu Asp Gly Ser Arg Val 20 25 30 Ala Trp Tyr Gln HisLys Pro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn ArgAla Ser Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala LeuTyr Tyr Cys Gln Met Tyr Gly Gly Phe Thr Tyr Thr 85 90 95 Phe Gly Gln GlyThr Lys Leu Glu Arg Lys Arg Thr 100 105 108 amino acids amino acidlinear protein not provided 85 Glu Leu Thr Gln Ser Pro Gly Thr Leu SerLeu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser Ser HisGln Leu Asp Gly Ser Arg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro Gly GlnAla Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg Ala Ser Gly IleSer Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr Cys GlnThr Tyr Gly Arg Gly Ser Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu GluArg Lys Arg Thr 100 105 108 amino acids amino acid linear protein notprovided 86 Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly GluArg 1 5 10 15 Ala Thr Phe Ser Cys Arg Ser Ser His Gln Leu Asp Gly SerArg Val 20 25 30 Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu ValIle His 35 40 45 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val GluPro Glu 65 70 75 80 Asp Phe Ala Leu Tyr Tyr Cys Gln Thr Tyr Gly Arg GlyHis Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100105 108 amino acids amino acid linear protein not provided 87 Glu LeuThr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 AlaThr Phe Ser Cys Arg Ser Ser His Gln Leu Asp Gly Ser Arg Val 20 25 30 AlaTrp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 GlyVal Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 GlySer Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80Asp Phe Ala Leu Tyr Tyr Cys Gln Thr Tyr Gly Arg Gly Ile Tyr Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr 100 105 108 amino acidsamino acid linear protein not provided 88 Glu Leu Thr Gln Ser Pro GlyThr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 15 Ala Thr Phe Ser Cys ArgSer Ser His Gln Leu Asp Gly Ser Arg Val 20 25 30 Ala Trp Tyr Gln His LysPro Gly Gln Ala Pro Arg Leu Val Ile His 35 40 45 Gly Val Ser Asn Arg AlaSer Gly Ile Ser Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp PheThr Leu Thr Ile Thr Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Leu TyrTyr Cys Gln Gln Tyr Gly Trp Pro Phe Tyr Thr 85 90 95 Phe Gly Gln Gly ThrLys Leu Glu Arg Lys Arg Thr 100 105 124 amino acids amino acid linearprotein not provided 89 Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro GlyAla Ser Val Lys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe SerHis Phe Thr Val His 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln Arg Phe GluTrp His Gly Trp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser AlaLys Phe Gln Asp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser Ala Asn ThrAla Tyr Met Glu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp Thr Ala ValTyr Tyr Cys Ala Arg Val 85 90 95 Gly Glu Trp Gly Trp Glu Met Phe Arg TyrAsp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Thr Val IleVal Ser Ser 115 120 124 amino acids amino acid linear protein notprovided 90 Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser ValLys 1 5 10 15 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser His Phe ThrLeu His 20 25 30 Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp His GlyTrp Ile 35 40 45 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe GlnAsp Arg 50 55 60 Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr MetGlu Leu 65 70 75 80 Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr CysAla Arg Val 85 90 95 Gly Glu Trp Gly Trp Glu Met Phe Arg Tyr Asp Asn TyrTyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser115 120 124 amino acids amino acid linear protein not provided 91 LeuGlu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser His Phe Thr Val His 20 25 30Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp His Gly Trp Ile 35 40 45Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 7580 Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 9095 Gly Glu Trp Gly Trp His Gln Arg Arg Tyr Asp Asn Tyr Tyr Met Asp 100105 110 Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 124amino acids amino acid linear protein not provided 92 Leu Glu Gln SerGly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 15 Val Ser CysGln Ala Ser Gly Tyr Arg Phe Ser His Phe Thr Leu His 20 25 30 Trp Val ArgGln Ala Pro Gly Gln Arg Phe Glu Trp His Gly Trp Ile 35 40 45 Asn Pro TyrAsn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gln Asp Arg 50 55 60 Val Thr PheThr Ala Asp Thr Ser Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 80 Arg SerLeu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 85 90 95 Gly GluTrp Gly Trp His Gln Arg Arg Tyr Asp Asn Tyr Tyr Met Asp 100 105 110 ValTrp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120

What is claimed is:
 1. A human monoclonal antibody mutagenized tocontain a complementary determining region that immunoreacts with andneutralizes human immunodeficiency virus-1 (HIV-1), wherein themonoclonal antibody reduces HIV-1 infectivity titer in an in vitro virusinfectivity assay by 50% at a concentration of from 5 to 100 nanograms(ng) of antibody per milliliter (ml).
 2. The human monoclonal antibodyof claim 1 wherein said concentration is less than 20 ng/ml.
 3. Thehuman monoclonal antibody of claim 1 wherein said concentration is lessthan 10 ng/ml.
 4. The human monoclonal antibody of claim 1 wherein saidHIV-1 is a first HIV-1 strain and wherein said monoclonal antibody hasthe capacity to reduce said HIV-1 infectivity titer of a second strainof HIV-1 by 50% at a concentration of less than 10 micrograms (ug) ofantibody per milliliter (ml).
 5. The human monoclonal antibody of claim1 wherein said antibody is a Fab fragment.
 6. The antibody of claim 1wherein the complementary determining region is in a light chainimmonoglobulin variable region.
 7. The antibody of claim 1 wherein thecomplementary determining region is in a heavy chain immunoglobulinvariable region.
 8. The human monoclonal antibody of claim 7 wherein theheavy chain immunoglobulin variable region comprises an amino acidresidue sequence having the sequence of SEQ ID NO 2, 3, 4 or
 5. 9. Thehuman monoclonal antibody of claim 1 that comprises an amino acidresidue sequences in pairs of SEQ ID NOs 2:6, 3:6, 4:6 or 5:6.
 10. Thehuman monoclonal antibody of claim 1 wherein said monoclonal antibodyimmunoreacts with HIV-1 gp120 with a dissociation constant (K_(d)) of1×10⁻⁸M or less.
 11. The human monoclonal antibody of claim 7 whereinthe heavy chain immunoglobulin variable region comprises an amino acidresidue sequence having the sequence of SEQ ID NOs 1, 3, 54, 55, 56, 57,58, 59, 89, 90, 91 or
 92. 12. The human monoclonal antibody of claim 6wherein the light chain immunoglobulin variable region comprises anamino acid residue sequence having the sequence of SEQ ID NOs 6, 69, 70,73, 75, 76, 77, 79, 80, 82, 83, 84, 85, 86, 87 or
 88. 13. The humanmonoclonal antibody of claim 10 wherein said dissociation constant isfrom 1×10⁻⁹M to 1×10¹⁰M.
 14. The human monoclonal antibody of claim 1that comprises at least one amino acid residue sequence in pairs of SEQID NOs 3:6, 3:69, 3:70, 3:73, 3:75, 3:76, 3:77, 3:79, 3:80, 3:82, 3:83,3:84, 3:87, 54:6, 55:6, 56:6, 57:6, 58:6, 59:6, 90:88, 91:6, 91:88 or92:88.
 15. The human monoclonal antibody of claim 10 wherein saiddissociation constant is from 1×10⁻¹⁰M to 1×10⁻¹¹M.
 16. The humanmonoclonal antibody of claim 10 wherein said dissociation constant isfrom 1×10⁻¹¹M to 1×10⁻¹²M.
 17. A polynucleotide sequence encoding aheavy chain immunoglobulin variable region amino acid residue sequenceof a mutagenized human monoclonal antibody that immunoreacts with humanimmunodeficiency virus-1 (HIV-1) glycoprotein gp120 and neutralizesHIV-1, wherein the heavy chain immunoglobulin variable region comprisesan amino acid residue sequence having the sequence of SEQ ID NOs 1, 2,3, 4, 5, 54, 55, 56, 57, 58, 59, 89, 90, 91 or 92, and polynucleotidesequences complementary thereto.
 18. A polynucleotide sequence encodinga light chain immunoglobulin variable region amino acid residue sequenceof a mutagenized human monoclonal antibody that immunoreacts with humanimmunodeficiency virus-1 (HIV-1) glycoprotein gp120 and neutralizesHIV-1, wherein the light chain immunoglobulin variable region comprisesan amino acid residue sequence having the sequence of SEQ ID Nos 6, 69,70, 73, 75, 76, 77, 79, 80, 82, 83, 84, 85, 86, 87 or 88, andpolynucleotide sequences complementary thereto.
 19. A polynucleotidesequence encoding a heavy and light chain immunoglobulin variable regionamino acid residue sequence of a mutagenized human monoclonal antibodythat immunoreacts with human immunodeficiency virus-1 (HIV-1)glycoprotein gp120 and neutralizes HIV-1, wherein the heavy and lightchain immunoglobulin variable regions comprise an amino acid residuesequence in pairs of SEQ ID NOs 2:6, 3:6, 4:6, 5:6, 3:69, 3:70, 3:73,3:75, 3:76, 3:77, 3:79, 3:80, 3:82, 3:83, 3:84, 3:85, 3:86, 3:87, 54:6,55:6, 56:6, 57:6, 58.6, 59:6, 89:6, 89:88, 90:86, 90:88, 91:6, 91:88 or92:88, and polynucleotide sequences complementary thereto.
 20. A hostcell comprising the polynucleotide sequence of claims 17, 18 or
 19. 21.A DNA expression vector comprising the polynucleotide sequence of claims17, 18 or
 19. 22. A method of detecting human immunodeficiency virus(HIV) comprising contacting a sample suspected of containing HIV with adiagnostically effective amount of the monoclonal antibody of claim 1and determining whether the monoclonal antibody immunoreacts with thesample.
 23. The method of claim 22, wherein the detecting is in vivo.24. The method of claim 23, wherein the monoclonal antibody isdetectably labelled with a label selected from the group consisting of aradioisotope and a paramagnetic label.
 25. The method of claim 22,wherein the detecting is in vitro.
 26. The method of claim 25, whereinthe monoclonal antibody is detectably labelled with a label selectedfrom the group consisting of a radioisotope, a fluorescent compound, acolloidal metal, a chemiluminescent compound, a bioluminescent compound,and an enzyme.
 27. The method of claim 25, wherein the monoclonalantibody is bound to a solid phase.
 28. A method for producing amutagenized human anti-HIV-1 monoclonal antibody comprising the stepsof: a) providing the genome of filamentous phage encoding a humanmonoclonal antibody having immunoglobulin heavy and light chain variabledomains, said heavy chain variable domain present as a fusionpolypeptide containing a filamentous phage membrane anchor domain,wherein said monoclonal antibody immunoreacts with HIV-1 glycoproteingp120; b) mutating the immunoglobulin heavy chain variable domain-codingnucleotide sequence present in the provided genome to form a firstlibrary of mutagenized phage particles containing a mutatedimmunoglobulin heavy chain variable domain nucleotide sequence; c)contacting the library formed in step (b) with a HIV-1 glycoproteingp120 ligand under conditions sufficient for members of the library tobind to the ligand and form a first ligand-phage particle complex; d)isolating phage particles in said first complex away from non-boundlibrary members to form a first ligand-enriched library comprising phageparticles having binding specificity for said HIV-1 glycoprotein gp120ligand; e) providing the genome of filamentous phage from said firstligand-enriched library; f) mutating the immunoglobulin heavy chainvariable domain-coding nucleotide sequence present in the providedgenome to form a second library of mutagenized phage particlescontaining a mutated immunoglobulin heavy chain variable domainnucleotide sequence; g) contacting the library formed in step (f) with aHIV-1 glycoprotein gp120 ligand under conditions sufficient for membersof the library to bind to the ligand and form a second ligand-phageparticle complex; and h) isolating phage particles in said secondcomplex away from non-bound library members to form a secondligand-enriched library comprising phage particles having bindingspecificity for said preselected HIV-1 ligand, thereby isolating asynthetic human monoclonal antibody immunoreactive with HIV-1.
 29. Themethod of claim 28 wherein said mutating in steps (b) and (f) aredirected to the same region of the immunoglobulin heavy chain variabledomain.
 30. The method of claim 28 wherein said mutating in steps (b)and (f) are directed to two different regions of the immunoglobulinheavy chain variable domain.
 31. The method of claim 28 wherein saidimmunoglobulin heavy chain variable domain is a complementaritydetermining region (CDR) selected from the group consisting of CDR1,CDR2 and CDR3.
 32. The method of claim 31 wherein said mutating in step(b) is directed to a first CDR and said mutating in step (f) is directedto a second CDR.
 33. The method of claim 32 wherein said first andsecond CDR's are CDR1 and CDR3, respectively.
 34. The method of claim 28wherein said mutating of step (b) comprises inducing mutagenesis in aCDR of an immunoglobulin gene in said genome which comprises amplifyinga portion of said CDR of the immunoglobulin gene by polymerase chainreaction (PCR) using a PCR primer oligonucleotide, said oligonucleotidehaving 5′ and 3′ termini and comprising: a) a nucleotide sequence atsaid 5′ terminus capable of hybridizing to a framework region upstreamof said CDR; b) a nucleotide sequence at said 3′ terminus capable ofhybridizing to a framework region downstream of said CDR; and c) anucleotide sequence between said 5′ and 3′ termini according to theformula: [NNS]_(n), wherein N is independently any nucleotide, S is G orC, and n is 3 to 24, said 3′ and 5′ terminal nucleotide sequences havinga length of 6 to 50 nucleotides, or an oligonucleotide having a sequencecomplementary thereto.
 35. The method of claim 34 wherein n is 5, saidCDR is CDR1, and said upstream and downstream framework regions are FR1and FR2, respectively.
 36. The method of claim 28 wherein said mutatingof step (f) comprises inducing mutagenesis in a CDR of an immunoglobulingene in said genome which comprises amplifying a portion of said CDR ofthe immunoglobulin gene by polymerase chain reaction (PCR) using a PCRprimer oligonucleotide, said oligonucleotide having 5′ and 3′ terminiand comprising: a) a nucleotide sequence at said 5′ terminus capable ofhybridizing to the antisense (noncoding) framework region downstream ofsaid CDR; b) a nucleotide sequence at said 3′ terminus capable ofhybridizing to the antisense (noncoding) framework region upstream ofsaid CDR; and c) a nucleotide sequence between said 5′ and 3′ terminiaccording to the formula: [MNN]_(n), wherein N is independently anynucleotide, M is A or C, and n is 3 to 24, said 3′ and 5′ terminalnucleotide sequences having a length of 6 to 50 nucleotides, or anoligonucleotide having a sequence complementary thereto.
 37. The methodof claim 36 wherein n is 4, said CDR is CDR3, and said upstream anddownstream framework regions are FR3 and FR4, respectively.
 38. Themethod of claim 28 wherein said second ligand-enriched library comprisesphage particles that contain synthetic antibody molecules that have thecapacity to reduce HIV-1 infectivity titer in an in vitro virusinfectivity assay by 50% at a concentration of less than 100 nanograms(ng) of antibody per milliliter (ml) of culture medium.
 39. A syntheticmonoclonal antibody produced by the method of claim
 38. 40. An antibodyproduced by the process of claim 28.